U.S. patent number 5,660,829 [Application Number 08/442,348] was granted by the patent office on 1997-08-26 for process for antibody directed enzyme prodrug therapy.
This patent grant is currently assigned to Cancer Research Campaign Technology, Zeneca Limited. Invention is credited to Philip John Burke, Robert Ian Dowell, Anthony Brian Mauger.
United States Patent |
5,660,829 |
Burke , et al. |
August 26, 1997 |
Process for antibody directed enzyme prodrug therapy
Abstract
Prodrugs, of generic formula I, are disclosed for use in
antibody directed enzyme prodrug therapy (ADEPT). The prodrugs are
substrates for carboxypeptidase G2 (CPG2) and yield more active
cytotoxic drugs than known products of CPG2 catalysed
reactions.
Inventors: |
Burke; Philip John (Charlton,
GB), Dowell; Robert Ian (Congleton, GB),
Mauger; Anthony Brian (Kensington, MD) |
Assignee: |
Zeneca Limited (London,
GB2)
Cancer Research Campaign Technology (London,
GB2)
|
Family
ID: |
26301303 |
Appl.
No.: |
08/442,348 |
Filed: |
May 16, 1995 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
361424 |
Dec 21, 1994 |
5587161 |
|
|
|
94952 |
Jul 22, 1993 |
5405990 |
Apr 11, 1995 |
|
|
Foreign Application Priority Data
|
|
|
|
|
Jul 23, 1992 [GB] |
|
|
9215636 |
May 26, 1993 [GB] |
|
|
9301884 |
|
Current U.S.
Class: |
424/178.1;
560/135; 514/476; 424/182.1; 560/137; 560/136; 560/134 |
Current CPC
Class: |
C07C
309/66 (20130101); A61K 47/6899 (20170801); C07C
311/51 (20130101); C07C 237/20 (20130101); B82Y
5/00 (20130101); A61P 35/00 (20180101); C07C
275/40 (20130101); A61P 43/00 (20180101); C07C
271/54 (20130101); C07D 257/04 (20130101); C07C
275/24 (20130101) |
Current International
Class: |
C07C
311/00 (20060101); C07C 311/51 (20060101); C07C
309/66 (20060101); C07C 237/20 (20060101); C07C
237/00 (20060101); C07C 309/00 (20060101); C07C
275/00 (20060101); C07C 275/24 (20060101); C07C
275/40 (20060101); C07C 271/54 (20060101); C07C
271/00 (20060101); A61K 47/48 (20060101); C07D
257/04 (20060101); C07D 257/00 (20060101); A61K
039/395 (); C07K 016/00 (); C07C 261/00 (); C07F
009/02 () |
Field of
Search: |
;560/134,135,136,137
;514/476 ;424/178.1,182.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
8807378 |
|
Oct 1988 |
|
WO |
|
8910140 |
|
Nov 1989 |
|
WO |
|
9002729 |
|
Mar 1990 |
|
WO |
|
9103460 |
|
Mar 1991 |
|
WO |
|
9308288 |
|
Apr 1993 |
|
WO |
|
Other References
Bagshawe, K.D., Tumour Site Activation of Cytotoxic Agent; Advances
in Applications of Monoclonal Antibodies in Clinical Oncology;
Hammersmith Hospital Meeting; 1988. .
Bagshawe, K.D., Antibody Directed Enzymes Acivate Anti-Cancer
Prodrugs Biochem Soc Trans; 1990. .
Parasmickiene, Izn. Akad. Nauk. SSSR Ser. Khim. (3) 649-51 (1971).
.
Karpavicius, Izv. Akad. Nauk. SSSR Ser. Khim (3) 2150-3 (1970).
.
CAS/STN-Search Report. .
G. Parasmickiene et al., `Synthesis of amino acid derivatives
acylated by p-[bis(2-chloropropyl)amino]phenyl alkanoic acids`
Chemical Abstracts, vol. 75, No. 5; Aug. 2, 1971, p. 564, Abstract
No. 36603m. .
K. Karpavicius et al., `N-p-[Bis(2-chloroethyl)amino]phenylacetyl
dicarboxylic amino acids and their derivatives` Chemical Abstracts,
vol. 75, No. 1; Jul. 5, 1971, p. 526, Abstract No. 6299z. .
M. H. Benn et al., `Cytotoxic Compounds. Part II. Some Amides of
the "Nitrogen Mustard" type.` Journal of the Chemical Society 1961;
pp. 2365-2375. .
P.D. Edwards et al., `Cytotoxic compounds Part XVII. o-,m-, and
p-Bis(2-chloroethylamino)phenol,p-[N-(2-chloroethyl
methylamino]phenol, N,N-[bis-2-chloroethyl-p-phenylenediamine], and
N,N-bis(2-chloroethyl)-N'-methyl-p-phe nylenediamine as sources of
biologically active carbamates`; Journal of the Chemical Society,
Perkin Transactions a, No. 20, 1973 pp. 2397, -2402. .
Edwards/Foster/Owen/Pringle: Cytotoxic Compounds XVII; Perkin
J.C.S., 1 (1973): 2397-2402. .
Benn/Creighton/Owen/White: Cytotoxic Compounds II (1961):
2365-2375. .
Wilman, D. E. V.: Prodrugs in Cancer Chemotheraphy: Biochem. Soc.
Trans. 14 (1986): 375-382. .
Springer, C.J., J. Med. Chem., 33, (1990), 677-681. .
Levy C.C., J. Bio. Chem., 212, (1967), 2933-2938. .
Pratt, A. G., J. Med. Chem., 243, (1968), 6367-6372. .
Levy, C.C., J. Bio Chem., 242, (1967), 2933-2938. .
McCullough, J.L., J. Bio Chem., 246, (1971), 7207-7213. .
Goldman, P., Proc. N.A.S., 58, (1967), 1299-1306. .
Rosowsky, A., Progress in Medicinal Chemistry, 26, (1990), 145-159.
.
Adamson P.C., J. Clinical Oncology, 10, (1992), 1359-1364. .
Bisset, G.M.F., J. Med. Chem., 35, (1992), 859-866. .
Sherwood, R.F., Eur. J. Biochem., 148 (1985), 447-453. .
Bagshawe K.D., Br. J. Cancer, 58, (1988) 700-703. .
Springer C.J., J. Med. Chem., 33 (1990), 677-681. .
Antoniw C.J., Br. J. Cancer, 62, (1990), 909-914. .
Mann, J., Tetrahedron, 46, (1990), 5377-5382. .
Springer, C.J., Eur J. Cancer, 27, (1991), 1361-1366. .
Bagshawe, K.D., Disese Markers, 9, (1991), 233-238. .
Springer, C. J., Anti-Cancer Drug Design, 6, (1991), 467-479. .
Springer, C. J., Drugs of the Future, 18(3), (1993), 212-215. .
Bagshawe, K.D., Monclonal Antibodies and Immunoconjugates, (1990),
95-102. .
Springer, C. J., Monoclonal Antibodies--Antibodies in Clinical
Oncology, (1991), 185-191. .
Bagshawe, K.D. Proc. 4th Intern. Conf. on Monoclonal Antibody
Imunoconjugates for Cancer, (1989), 178. .
Sharma, S. K., Disease Markers, 9 (1991), 225-231. .
Springer, C. J. Proc. Advances in the Applications of Monoclonal
Antibodies in Clinical Oncology, RPMS, UK 34, (1989). .
Bagshawe, K.D., Biol. Proc. 17th Intern. Soc. Oncodevelopmental
Biology & Medicine, DEX(1989). .
Melton, R. G., Proc. NATO Adv. Studies Inst., Greece (1994). .
Antoniw, P., Proc., 18th Intern. Soc. Oncodevelopment Biology &
Medicine, Russia (1990). .
Sharma, S. K. Proc. 18th Intern. Soc. Oncodevelopment Biology &
Medicine, Russia (1990). .
Springer, C. J., Proc. Adv. in Applications of Monoclonal
Antibodies in Clinical Oncology RPMS, UK 23 (1990). .
Sunters, C. J. Biochem. Pharmacology, 44(1), (1992), 59-64. .
Blakely, D. C., Proc. 9th Intern. Hammersmith Meeting, Porto
Carras, Greece 33, (1992). .
Springer, C. J., Proc. 9th Intern. Hammersmith Meeting, Advances in
Applications of Monoclonal Antibodies in Clinical Oncology, Porto
Carras, Greece 35 (1992). .
Sunters, A., Br. J. Cancer, 60 (1992). .
Blakey, D. C., Br. J. Cancer, 67 (1992). .
Sharma, S. K., Proc. Advances in the Applications of Monoclonal
Antibodies in Clinical Oncology, London, UK 53 (1989). .
Sharma, S. K. Antib. Immunoconj. & Radiopharm. 6 (1993), 74.
.
Sharma, S. K., Proc. 10th Intern. Hammersmith Meeting--Advances in
Applications of Monoclonal Antibodies in Clinical Oncology, Paphos,
Cyprus, (1993), 24-25. .
Bagshawe, K. D., Antib. Immunoconj. & Radiopharm, (1992), 133.
.
Springer, C. J., Antib. Immunoconj. & Radiopharm., (1991), 226.
.
Bagshawe, K. D., Antib. Immunoconj. & Radiopharm, (1991), 204.
.
Springer, C. J., Antib. Immunoconj. & Radiopharm., (1992), 127.
.
Bagshawe, K. D., Antib. Immunoconj. & Radiopharm., 4 (1991),
915-922. .
Springer, C. J., Proc. 3rd Intern. Conf. on Monoclonal Antibody
Immunoconjugates for Cancer, UCSD, USA (1988), 43. .
Springer, C. J., Antib. Immunoconj. & Radiopharm. 3 (1990), 61.
.
Bagshawe, K. D., Proc. 4th Int. Conf. on Monoclonal Antibody
Immunoconjugates for Cancer, USCD, USA (1989) 178. .
Sharma, S K., Antib. Immunoconj. & Radiopharm., 5, (1992) 348.
.
Springer, C. J., Antib. Immunoconj. & Radiopharm., 6 (1993),
74. .
Springer, C. J., Comparasion of Half Lives and Cytotoxicity of
Prodrugs & their Activated Drugs in Antibody Directed Enzyme
Prodrug Therapy (Adept); Proc. Targeted Cancer Therapy, London,
1991. .
Springer, C. J., Proc 10th Int. Hammersmith Meeting--Advances in
Application of Monoclonal Antibodies in Clinical Oncology, Cyprus
(1993). .
Bagshawe, K.D., (1990) Proc. Advances in Applications of Monoclonal
Antibodies in clerical Oncology, London, U.K. .
Sharma et al., (1990) Antibody directed enzyme prodrug therapy
(ADEPT) in human tumour xenograft models Br. J. Cancer, 62;
487..
|
Primary Examiner: Achutamurthy; Ponnathapura
Attorney, Agent or Firm: Cushman Darby & Cushman
Intellectual Property Group of Pillsbury Madison & Sutro,
LLP
Parent Case Text
This is a continuation of application Ser. No. 08/361,424 filed
Dec. 21, 1994, now U.S. Pat. No. 5,587,161, which is a divisional
of application Ser. No. 08/094,952 filed Jul. 23, 1993, now U.S.
Pat. No. 5,405,990 issued Apr. 11, 1995.
Claims
We claim:
1. A process for the preparation of
N-(4[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic
acid-.gamma.-(3,5-dicarboxy)anilide which comprises deprotecting a
compound of the formula:
wherein Pr1 and Pr2, which may be the same or different, represent
carboxyl groups in protected form.
2. The process of claim 1 wherein Pr1 and Pr2 are --COOBz.
3. The process of claim 1 wherein said deprotecting is carried out
by hydrogenation.
4. A method for the delivery of a cytotoxic drug to a site which
comprises administering to a host a first component, which first
component comprises an antibody or fragment thereof capable of
binding a given antigen present at said site, the antibody or
fragment thereof being conjugated to a carboxypeptidase G enzyme
capable of converting the compound
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic
acid-.gamma.-(3,5-dicarboxy)anilide or a physiologically acceptable
salt thereof into a cytotoxic drug; followed by administration of
the host of a second component, which second component comprises
the compound
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic
acid-.gamma.-(3,5-dicarboxy)anilide or a physiologically acceptable
salt thereof convertible under the influence of the
carboxypeptidase G enzyme to a cytotoxic drug, whereby at least a
portion of said compound is converted to a cytotoxic drug at said
site.
5. A method according to claim 4 wherein the first component is
further defined as a F(ab').sub.2 fragment of antibody A5B7
conjugated to carboxypeptidase G2.
Description
The invention relates to compounds for use in antibody directed
enzyme prodrug therapy (ADEPT), processes for their preparation,
pharmaceutical compositions containing them and methods for their
use as well as to a two component system comprising 1) a conjugate
of an enzyme and an antibody or antibody fragment and 2) a compound
of the present invention. The compounds are particularly of
interest at pro-drugs for use in conjunction with carboxypeptidase
G enzymes, particularly carboxypeptidase G2 (CPG2).
Many cytotoxic compounds have been discovered which are of
potential use in cancer chemotherapy. Nitrogen mustards form one
important family of such cytotoxic compounds. The clinical use of
cytotoxic compounds in general and nitrogen mustards in particular
has been limited because of the poor selectivity in the cytotoxic
effect between tumour cells and normal cells.
One approach to overcome this problem has involved the development
of so-called pro-drugs which are derivatives of the cytotoxic drug,
often a relatively simple derivative, whose cytotoxic properties
are considerably reduced compared to those of the parent drug.
Proposals have been made for the administration of such pro-drugs
to patients under regimes whereby the pro-drug is only converted to
the cytotoxic drug in the region of the intended site of
action.
One approach involves linkage of a cytotoxic parent nitrogen
mustard with an amino acid to form a pro-drug which can be
converted to the parent nitrogen mustard at the site of intended
action under the influence of an enzyme. This approach can be put
into practise by the utilisation of an antibody/enzyme conjugate in
association with a pro-drug. The antibody/enzyme conjugate is
formed from an antibody selective for tumours and an enzyme that
will convert the pro-drug to the cytotoxic drug. In clinical
practice, the antibody/enzyme conjugate is first administered to
the patient and is allowed to bind to the tumour. After a suitable
period of time, to allow clearance of the antibody/enzyme conjugate
from the rest of the body, the pro-drug is administered to the
patient. Conversion of the pro-drug, under the influence of the
localised enzyme, to the cytotoxic drug takes place mainly in the
region of the tumour. Such a system is described in International
Application PCT/GB88/00181 published as WO88/07378 and U.S. Pat.
No. 4,975,278.
Known pro-drugs for ADEPT cleaved by CPG yield benzoic acid
mustards as their active drugs. However there is a need for more
active drugs to be produced by CPG cleavage to increase the
therapeutic potency against tumour cells. A further need arises to
increase the selectivity of ADEPT therapy with CPG, that is to say
the ratio of toxicity to cancer Cells compared with healthy
cells.
The present invention is based on the discovery of novel pro-drugs
for use in ADEPT therapy that are cleaved by CPG and which yield
significantly more active cytotoxic drugs than known products of
CPG catalysed reactions. CPG acts naturally as a folate degrading
enzyme which specifically hydrolyses glutamic and aspartic acids
from folate derivatives (Sherwood, R. F. et al., Eur. J. Biochem.
(1985), 148, 447-453). Carboxypeptidase G enzymes do not recognise
non-classical folate analogues (Kalghatgi, K. K. et al., Cancer
Research (1979), 39, 3441-3445) and are therefore considered
conservative in substrate specificity. Surprisingly the pro-drugs
of the present invention are substrates for CPG enzymes such as
CPG1, but particularly for CPG2 enzymes. CPG2 is an exopeptidase
with specificity for L-glutamate. It is known to hydrolyse the
glutamic acid moiety from folic acid and analogues thereof and
glutamyl-p-aminobenzoic acid by cleavage at --CO--NH-- of the
partial structure -aromatic ring--CO--NH--Glu. In the present
invention a partial structure for comparative purposes is
-aromatic-ring--X--CO--NH--Glu wherein X is --NH--, --O-- or
--CH2--; thus altering the distance between the cleavage point and
the aromatic ring as well as altering the electron distribution
across the --CO--NH-- bond, particularly when X is NH or O. CPG2
could not be predicted to accommodate these spatial and electronic
differences.
According to one feature of the present invention there are
provided compounds of Formula I, which are pro-drug substrates for
CPG enzymes, ##STR1## wherein R.sup.1 and R.sup.2 each
independently represents chlorine, bromine, iodine, OSO.sub.2 Me,
or OSO.sub.2 phenyl (wherein phenyl is optionally substituted with
1,2,3,4 or 5 substituents independently selected from
C.sub.1-4 alkyl, halogen, --CN or --NO.sub.2);
R.sup.1a and R.sup.2a each independently represents hydrogen,
C.sub.1-4 alkyl or C.sub.1-4 haloalkyl;
R.sup.3 and R.sup.4 each independently represents hydrogen,
C.sub.1-4 alkyl or C.sub.1-4 haloalkyl;
R5a, R5b, R5c and R5d each independently represents hydrogen,
C.sub.1-4 alkyl optionally containing one double bond or one triple
bond, C.sub.1-4 alkoxy, halogen, cyano, --NH.sub.2, --CONR.sup.7
R.sup.8 (wherein R.sup.7 and R.sup.8 are as defined below),
--NH(C.sub.1-4 -alkyl), --N(C.sub.1-4 -alkyl).sub.2 and C.sub.2-5
alkanoyl; or
R5a, and R5b together represent
a) C4 alkylene optionally having one double bond;
b) C3 alkylene; or
c) --CH.dbd.CH--CH.dbd.CH--, --CH.dbd.CH--CH2-- or
--CH2--CH.dbd.CH-- each optionally substituted with 1, 2, 3 or 4
substituents said substituents each independently selected from the
group consisting of C.sub.1-4 alkyl, C.sub.1-4 alkoxy, halogen,
cyano, nitro, C.sub.2-5 alkanoyl and --CONR7R8 (wherein R7 and R8
are as defined below);
X represents O, NH or --CH.sub.2 --;
Y represents O;
Z represents --V--W where V is --CH2--T-- in which T is --CH2--,
--O--, --S--, --(SO)-- or --(SO.sub.2)-- (provided that when V has
sulphur or oxygen as its second atom, W is other than --COOH) and
said group V optionally further carrying one or two substituents Q1
and/or Q2 on carbon; wherein Q.sup.1 and Q.sup.2 each independently
represents C.sub.1-4 alkyl or halogen; or, when Q1 and Q2 are
bonded to adjacent carbon atoms, Q.sup.1 and Q.sup.2 together may
additionally represent a C.sub.3 -C.sub.4 alkylene radical
optionally substituted with 1, 2, 3 or 4 substituents independently
selected from the group consisting of C.sub.1-4 alkyl and halogen;
and
W represents
(1) COOH,
(2) --(C.dbd.O)--O--R6 wherein R6 represents a C.sub.1-6 alkyl,
C.sub.3-6 cycloalkyl or aryl (as defined in 3 below) group;
(3) --(C.dbd.O)--NR7R8 wherein R7 and R8 each independently
represent hydrogen or a C1-6alkyl, C3-6cycloalkyl, aryl, heteroaryl
linked to N via carbon or C7-9aralkyl group wherein
aryl is phenyl;
heteroaryl is a 5 or 6 membered ring containing 1 to 3 heteroatoms
selected from the group consisting of nitrogen and sulphur;
the aryl moiety per se, the heteroaryl moiety and the aryl moiety
of the aralkyl group may be substituted on carbon with 1-4
substituents selected from the group consisting of --COOH, --OH,
--NH.sub.2, --CH.sub.2 --NH.sub.2, --(CH2).sub.1-4 --COOH,
tetrazol-5-yl and --SO.sub.3 H and the alkyl moiety may optionally
carry a methyl group;
(4) --SO.sub.2 NHR9 wherein R9 is as defined for R7 but may
additionally represent --CF.sub.3, --CH.sub.2 CF.sub.3 or aryl as
defined above;
(5) SO.sub.3 R10 in which R10 represents H, C.sub.1-6 alkyl or
C.sub.3-6 cycloalkyl,
(6) PO.sub.3 R10R10 (wherein the R10 radicals, which may be the
same or different, are as herein defined)
(7) a tetrazol-5-yl group;
(8) --CONH--SO.sub.2 R11 in which R11 represents
(a) C.sub.3-7 cycloalkyl;
(b) C.sub.1-6 -alkyl optionally substituted with substituents
selected from the group consisting of aryl as defined below,
C.sub.1-4 -alkyl, CF.sub.3 or halogen; and
(c) perfluoro-C.sub.1-6 alkyl; wherein aryl is phenyl or phenyl
having 1-5 substituents wherein the substituents are selected from
the group consisting of halogen, --NO.sub.2, --CF.sub.3, C.sub.1-4
alkyl, C.sub.1-4 alkoxy, --NH.sub.2, --NHCOCH.sub.3, --CONH.sub.2,
--OCH.sub.2 COOH, --NH(C.sub.1-4 -alkyl), --N(C.sub.1-4
-alkyl).sub.2, --NHCOOC.sub.1-4 alkyl, --OH, --COOH, --CN and
--COOC.sub.1-4 alkyl; and
(9)--M--Het wherein M represents S, SO or SO2 and Het represents a
5 or 6 membered heterocyclic aromatic ring linked to M via a carbon
atom of the aromatic ring, said aromatic ring containing 1, 2, 3 or
4 heteroatoms selected from the group consisting of O, N and S said
aromatic ring optionally being substituted on carbon atoms of the
ring with 1, 2, 3 or 4 substituents selected from the group
consisting of --OH, --SH, --CN, --CF3, NH2 and halogen; and salts
of said compound of formula I.
The compounds of formula I possess at least one asymmetric carbon
atom, that being the carbon atom carrying the substituent --COOH in
formula I. Moreover, depending on the meanings of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, Q.sup.1 and Q.sup.2, the compounds of formula I
may carry additional asymmetric carbon atoms. It will be understood
that the present invention encompasses all such forms of the
compound of formula I, including the racemic form as well as the
individual optical isomers thereof which possess the useful
physiological properties of the compositions of the present
invention defined herein, it being common general knowledge to
those skilled in the art how such isomers maybe separated and how
their physiological properties may be determined. Compounds of the
present invention preferably possess an L configuration at the
carbon atom carrying the substituent --COOH in Formula I.
The present invention encompasses the salts of the compounds of
formula I. It will be appreciated, however, that for pharmaceutical
use, the salts referred to will be pharmaceutically acceptable, but
other salts may find use, for example in the preparation of
compounds of formula I and their pharmaceutically acceptable salts.
Polymorphic forms of compounds of the present invention may be
prepared and these forms are also encompassed by the invention.
Where any substituent referred to herein represents or contains an
alkyl group, such group may be straight chain or branched. Where
any substituent referred to herein represents or contains a
C.sub.1-6 alkyl group such group advantageously has 1 to 4 carbon
atoms, for example methyl, ethyl, n-propyl or isopropyl, preferably
methyl and ethyl, but especially methyl. Where any substituent
referred to herein represents or contains a C.sub.1-4 alkyl group
such group may for example be methyl, ethyl, n-propyl or isopropyl,
preferably methyl and ethyl, but especially methyl.
Preferred values for R.sup.1 and R.sup.2 are I, Br, Cl, OSO2Me and
OSO.sub.2 phenyl wherein phenyl is substituted with 1 or 2
(especially 1) substituents (as herein defined) in the 2 and/or 4
positions. Especially preferred values for R.sup.1 and R.sup.2 are
I, Br, Cl and --OSO.sub.2 Me. A preferred value for R.sup.1a and
R.sup.2a is --CH3 or hydrogen, but especially hydrogen.
Preferred values for R.sup.3 and R.sup.4 are hydrogen, methyl and
CF.sub.3, but especially hydrogen.
Preferred values for R5a-d are hydrogen, fluorine, chlorine,
methyl, --CONH.sub.2 and CN. Where substitution is present on the
phenyl ring such that at least one of R.sup.5a, R.sup.5b, R.sup.5c
and R.sup.5d is other than hydrogen it is preferred that only one
or two of R.sup.5a, R.sup.5b, R.sup.5c and R.sup.5d are other than
hydrogen. In such circumstances it is further preferred that
R.sup.5b and R.sup.5d are hydrogen and that R.sup.5a and/or
R.sup.5c is other than hydrogen. It is especially preferred however
that R.sup.5a-b are hydrogen.
Preferred values for X are O or N, especially O. In another
embodiment of the invention an especially preferred value for X is
N.
A preferred value for V is --CH2--CH2--. In another embodiment of
the invention a preferred value for V, when W is tetrazol-5-yl, is
--CH2--S--.
Where W represents a group of definition 3, aryl is preferably
substituted phenyl and heteroaryl is advantageously a 5 or 6
membered ring containing 1 or 2 heteroatoms, such heteroatoms
preferably being nitrogen such as pyridyl or pyrimidinyl. Preferred
substituents on the aryl moiety per se, the heteroaryl moiety or
the aryl moiety of the aralkyl group are --COOH, --CH.sub.2 --COOH
or tetrazol-5-yl.
Where W represents a group of definition 8(b) in which W represents
a C.sub.1-6 alkyl group optionally substituted by aryl, the
optionally substituted aryl group is preferably phenyl substituted
by --CONH.sub.2, --OCH.sub.2 --COOH and/or --COOH, but is
especially unsubstituted phenyl.
Where W represents a group of definition 9, Het advantageously
represents a 5- or 6- membered heterocyclic aromatic ring
containing 1, 2, 3 or 4 nitrogen atoms. Preferably nitrogen is the
only heteroatom present in the ring. Particular groups thus include
pyridyl, pyrrolyl, 1,2,3-triazinyl and 1,2,4-triazinyl.
Particular values for W are the definitions 1, 2, 3, 5, 6, 7 and 9,
preferred definitions being 1, 2, 3, 7 and 9 as detailed
hereinbefore.
More particular values for W are
--COOH,
--CONH.sub.2,
CONHR8 (wherein R8 is as hereinbefore defined, especially wherein
R8 is phenyl),
tetrazol-5-yl,
--CONH--SO.sub.2 R.sup.11 (wherein R.sup.11 represents definitions
(b) and (c) as detailed hereinbefore) and the group
--M--Het (in which M is S and Het represents a 5-membered
heterocyclic aromatic ring with 3 or 4 heteroatoms optionally
substituted on carbon where the heterocyclic ring has 3-heteroatoms
by a halogen atom or cyano group).
Where W represents --CONH--SO.sub.2 R.sup.11 in which R.sup.11
represents a perfluoro-C.sub.1-6 alkyl group the perfluoroalkyl
group preferably has 1 to 4 carbon atoms, especially 1 or 2 carbon
atoms.
More particular values for W are --COOH, tetrazol-5-yl or
--CONH(aryl) (in which aryl is defined in definition 3 above). In
this context, aryl is preferably substituted phenyl, preferred
substituents being --COOH, --CH.sub.2 --COOH or tetrazol-5-yl.
Preferred specific compounds of the invention, by virtue of their
utility in ADEPT include:
(S)-2-(4-[bis(2-chloroethyl)amino]phenoxycarbonylamino)-4-(1H-1,2,3,4-tetra
zol-5-yl)butyric acid and salts thereof;
N-(4-[bis(2-chloroethyl)amino]-3-fluorophenylcarbamoyl)-L-glutamic
acid and salts thereof;
N-(4-[bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid and
salts thereof;
but an especially preferred compound of the invention is
N-(4-[bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic acid and
salts thereof.
Another preferred compound, which has shown good activity in tests,
is N-(4-[bis(2-iodoethyl)amino]phenoxycarbonyl)-L-glutamic acid and
salts thereof.
Particular sub-groups of the compounds of the present invention of
interest may be obtained by taking any one of the above mentioned
particular or generic definitions for R.sup.1 -R.sup.4, R.sup.5a-d,
X, Y, W, Q1 or Q2 either singly or in combination with any other
particular or generic definition for R.sup.1 -R.sup.4, R.sup.5a-d,
X, Y, W, Q1 or Q2.
Furthermore drugs produced by CPG cleavage of tested compounds of
the present invention for effective ADEPT are less stable under
physiological conditions than known drug products of CPG2 catalysed
reactions. This reduced stability produces less toxicity to healthy
cells, than if the drugs had stabilities similar to known products
of CPG catalysed reactions for ADEPT, if a proportion of active
drug produced at the tumour site leaks into the general
circulation. Thus tests we have conducted demonstrate that even
after intravenous administration of drug (that is active drug, not
pro-drug), 15 min later, the drug was not detectable in plasma.
The compounds of this invention form salts with various inorganic
and organic acids and bases and such salts are within the scope of
the invention. Such salts include ammonium salts, alkali metal
salts like sodium and potassium salts, alkaline earth metal salts
like the calcium and magnesium salts, salts with organic bases;
e.g. dicyclohexylamine salts, N-methyl-D-glucamine, salts with
amino acids like arginine, lysine, and the like. Also, salts with
organic and inorganic acids may be prepared; e.g., HCl, HBr,
H.sub.2 SO.sub.4, H.sub.3 PO.sub.4, methanesulfonic,
toluenesulfonic, and camphorsulfonic. Preferably the acids are
strong acids having pK.sub.a values less than or equal to 2 and
especially having pKa values less than or equal to 1.
Physiologically acceptable salts are preferred, although other
salts may be useful; e.g., in isolating or purifying the
product.
The salts can be formed by conventional means such as by reacting
the free acid or free base forms of the product with one or more
equivalents or the appropriate base or acid in a solvent or medium
in which the salt is insoluble, or in a solvent such as water which
is then removed in vacuo or by freeze-drying or by exchanging the
cartons of an existing salt for another cation on a suitable ion
exchange resin.
According to a further feature of the present invention there is
provided a process for the preparation of compounds of formula I
and salts thereof which comprises deprotecting a compound of the
formula: ##STR2## (wherein R.sup.1, R.sup.2, R.sup.1a, R.sup.2a,
R.sup.3, R.sup.4, R.sup.5a, R.sup.5b, R.sup.5c, R.sup.5d, X, Y,
Q.sup.1 and Q.sup.2 are as hereinbefore defined and Z.sup.1
represents Z as hereinbefore defined with the proviso that when W
is a carboxyl group it is present in protected form (denoted
Pr.sup.2) and Pr.sup.1 also represents a carboxyl group in
protected form (which may be the same or different to Pr.sup.2) and
if desired converting a compound of formula I thus obtained into a
salt thereof.
Pr.sup.1 and Pr.sup.2 may thus for example represent
benzyloxycarbonyl groups, t-butyloxy carbonyl groups,
2-(trimethylsilyl)ethyl ester, dimethyl-tert-butylsilyl ester,
diphenylmethyl ester, tetrahydropyran ester, tetrahydrofuran ester,
methoxyethoxymethyl ester or benzyloxymethyl ester or ether
generally known carboxy protecting groups, for example ester
forming protecting groups, for deprotection by hydrogenolysis or
acid catalysis (Greene, T. W. and Wuts, P. G. M. in Protective
Groups in Organic Synthesis, 2nd Edition, Wiley-Interscience,
1990).
Where Pr.sup.1 and/or Pr.sup.2 represents a benzyloxycarbonyl
group, deprotection is preferably effected by hydrogenation. Such
hydrogenation may be effected by any convenient means such as in
the presence of platinum or Raney nickel, but is preferably
effected by the use of palladium in the presence of carbon. The
hydrogenation is advantageously effected in the presence of an
inert solvent, preferably a non-protonic solvent, especially ethyl
acetate, tetrahydrofuran or polar aprotic solvent such as
dimethylformamide; preferably at a temperature of
0.degree.-100.degree. C. more preferably at a temperature of
15.degree.-50.degree. C. and especially at ambient temperature; and
preferably for 1-24 h.
Where Pr.sup.1 and/or Pr.sup.2 represents a t-butyloxycarbonyl
group, the deprotection reaction may advantageously be effected in
the presence of an acid, advantageously a strong acid such as
trifluoroacetic acid HCl, HBr, HI or formic acid. Where it is
desired to use a solvent, inert non protonic solvents such as
CH.sub.2 Cl.sub.2 or diethyl ether are preferred. The reaction is
conveniently effected at a temperature of 0.degree.-100.degree. C.,
more conveniently at 0.degree.-30.degree. C. and especially at
ambient temperature.
The compounds of formula Ia (as hereinbefore defined) and the salts
thereof are novel and thus constitute a further feature of the
present invention.
According to a further feature of the present invention there is
provided a process for the preparation of compounds of formula Ia,
wherein X is 0 and Y is 0, and the salts thereof which process
comprises reacting a compound of the formula: ##STR3## (wherein
R.sup.1, R.sup.2, R.sup.1a, R.sup.2a, R.sup.3, R.sup.4, R.sup.5a,
R.sup.5b, R.sup.5c and R.sup.5d are as hereinbefore defined X is 0,
Y is 0 and L represents a leaving atom or group) with a compound of
the formula: ##STR4## (wherein Pr.sup.1 and Z.sup.1 are as
hereinbefore defined) whereby to form a compound of formula Ia,
and, if desired, converting the compound of formula Ia to a salt
thereof. Advantageously L is Cl, Br, I, 4-nitrophenoxy or
pentafluorophenoxy. Conveniently the reaction is effected in the
presence of a solvent at a temperature of -10.degree. C. to
100.degree. C. and preferably at 20.degree.-50.degree. C.
Preferred reaction conditions include effecting the reaction in the
presence of an organic solvent (especially chloroform, ethyl
acetate, toluene, dimethylformamide and CH.sub.2 Cl.sub.2)
preferably at 5.degree.-50.degree. C. preferably for 1-24 h.
Compounds of formula (II) constitute a further feature of the
present invention which compounds may for example be prepared by
reacting a corresponding phenol containing an amino mustard group
either with an aryl chloroformate, for example nitrophenyl
chloroformate (especially 4-nitrophenylchloroformate) or with
phosgene whereby to form a compound of formula (II).
Preferred reaction conditions include effecting the reaction in the
presence of an organic solvent (especially ethyl acetate or
chloroform), preferably at 15.degree.-50.degree. C. (especially at
ambient temperature), preferably for 1-10 h.
Compounds of formula (III) wherein W represents group (3) as herein
defined may be prepared from compounds of formula (III) wherein W
represents group (2) as herein defined using standard conditions.
Preferable standard conditions include reaction with nitrogen
nucleophiles (especially ammonia or a primary or secondary amine)
preferably at 25.degree.-50.degree. C. preferably for about 24
h.
Compounds of formula (III) wherein W represents a tetrazol-5-yl
group may be prepared from the corresponding nitrile by known
methods for example that described by Finnegan, W G et al., JACS,
80, 1978, 3909 followed by deprotection of the amine (Pr.sup.3)
whereby to form compounds of formula (III) wherein W is
tetrazol-5-yl.
Compounds of formula (III) wherein W represents group (9) as herein
defined may be prepared by reduction of compounds of formula (XV)
as herein defined to a corresponding primary alcohol using standard
conditions (especially using diborane, or a mixed anhydride
reaction followed by sodium borohydride reduction). The
corresponding primary alcohol is converted by standard methods for
example using methane sulphonyl chloride and trimethylamine at
0.degree. C. in the presence of CH.sub.2 Cl.sub.2 into a leaving
group such as Br, I or mesylate; which leaving group is displaced
by a group of formula:
(wherein Het is as hereinbefore defined for W=(9)) whereby, after
treatment under oxidizing conditions, to form compounds of formula:
##STR5## (wherein Z.sup.111 represents Z as hereinbefore defined
with the proviso that W represents --S--Het, --(S.dbd.O)--Het or
--SO.sub.2 --Het, and Pr.sup.1 and Pr.sup.3 are protected carboxyl
and --NH.sub.2 groups respectively as hereinbefore defined).
Suitable oxidising conditions include treatment with an oxidising
agent (especially 3-chloroperbenzoic acid). Deprotection of the
--NH.sub.2 group of compounds of formula (XVII) under standard
conditions as hereinbefore defined is effective to form a compound
of formula (III) wherein W represents group (9) as hereinbefore
defined.
According to a further feature of the present invention there is
provided a process for the preparation of compounds of formula Ia,
wherein X is N and Y is 0, and the salts thereof, which process
comprises reacting a compound of the formula: ##STR6## (wherein
R.sup.1, R.sup.2, R.sup.1a, R.sup.2a, R.sup.3, R.sup.4 and
R.sup.5a-d are as hereinbefore defined) or a salt thereof with a
compound of the formula (III) as hereinbefore defined whereby to
form a compound of formula Ia, and, if desired, converting the
compound of formula Ia to a salt thereof. The reaction may for
example be effected in the presence of an organic solvent
(preferably polar aprotic, especially CH.sub.2 Cl.sub.2 or ethyl
acetate) preferably at room temperature preferably for about 1-5
hour.
According to a further feature of the present invention there is
provided a process for the preparation of compounds of formula (IV)
and the salts thereof, which process comprises reacting a compound
of formula (V) (wherein R.sup.1, R.sup.2, R.sup.1a, R.sup.2a,
R.sup.3, R.sup.4, and R.sup.5a-d are as hereinbefore defined) with
a compound of formula L.sup.1 --(C.dbd.O)--L.sup.2 (wherein L.sup.1
and L.sup.2 represent leaving groups) whereby to form a compound of
formula (IV). Values for L.sup.1 and L.sup.2 include Cl, CCl.sub.3,
imidazolyl and aryloxy (especially phenoxy). Preferred reaction
conditions include effecting the reaction in the presence of an
organic solvent (preferably polar aprotic, especially ethyl
acetate) at 5.degree.-25.degree. C. for about 15 min. ##STR7##
According to a further feature of the present invention there is
provided a process for the preparation of compounds of formula (Ia)
as hereinbefore defined, wherein X is --CH.sub.2 -- and Y is 0, and
the salts thereof which process comprises: reacting a compound of
formula (II) or a salt thereof, as hereinbefore defined, with the
provisos that X is --CH.sub.2 --, Y is 0 and L is advantageously
pentafluorophenoxy, Cl, --O--(CO)--C.sub.1-6 alkyl preferably
branched alkyl, especially C.sub.1-4 alkyl and the product of a
corresponding phenyl acetic acid containing an anilino mustard
reacted with a carbodiimide (especially dicyclohexylcarbodiimide);
with a compound of formula (III) as hereinbefore defined using
standard reaction conditions (for example effecting the reaction in
the presence of polar aprotic solvents such as dimethylformamide,
ethyl acetate or tetrahydrofuran for 1-24 h at
20.degree.-50.degree. C.) whereby to form a compound of formula
(Ia), and, if desired, converting the compound of formula (Ia) to a
salt thereof. Compounds of formula (II) may be prepared using
standard methods from corresponding phenyl acetic acids containing
an anilino mustard group.
According to a further aspect of the present invention there is
provided a process for the preparation of a compound of formula
(Ia) as hereinbefore defined, wherein W represents group (8) (that
is --CONH--SO.sub.2 R.sup.11) as hereinbefore defined or a salt
thereof which process comprises: reacting a compound of formula
(III) as hereinbefore defined with the proviso that W only
represents group (8); with a compound of formula (II) as
hereinbefore defined using reaction conditions known per se whereby
to form a compound of formula (Ia) wherein W represents group (8),
and, if desired, converting the compound of formula (Ia) into a
salt thereof. Preferred reaction conditions include effecting the
reaction in the presence of an organic solvent (preferably a polar
aprotic solvent, especially ethyl acetate or dichloromethane),
preferably at 5.degree.-50.degree. C. (especially ambient
temperature), preferably for 1-5 h.
Compounds of formula (III) constitute a further aspect of the
present invention and such compounds may be prepared by
deprotection of the amine group .of a compound of formula: ##STR8##
(in which Pr.sup.1 represents a carboxyl group in protected form as
hereinbefore defined, Pr.sup.3 represents a --NH.sub.2 group in
protected form, especially a benzyloxycarbonyl derivative or a
phthalimido derivative and Z.sup.1 represents Z as hereinbefore
defined with the proviso that W represents group (8) under reaction
conditions known per se whereby to form a compound of formula (III)
wherein W represents group (8) and, if desired, converting the
compound of formula (III) into a salt thereof. Preferred reaction
conditions include hydrogenation in the presence of palladium on
carbon in an organic solvent (preferably polar aprotic, especially
ethyl acetate or tetrahydrofuran), preferably at ambient
temperature, preferably for 1-24 h. Further preferred reaction
conditions include effecting deprotection in the presence of HBr in
acetic acid, preferably at ambient temperature, preferably for 1-24
h.
Compounds of formula (XI) may be prepared by reacting a compound of
formula: ##STR9## (wherein Pr.sup.1 and Pr.sup.3 are as
hereinbefore defined and Z.sup.11 represents Z as hereinbefore
defined with the proviso that W represents only COOH); with a
compound of formula R.sup.11 SO.sub.2 NH.sub.2 in which R.sup.11 is
as defined in W=(8) whereby to form a compound of formula (XI)
under standard reaction conditions. Standard reaction conditions
include carrying out the reaction in an inert solvent (especially
dichloromethane) in the presence of a carbodiimide (especially
dicyclohexylcarbodiimide) and a base (especially
4-(N,N-dimethylamino)piperidine).
According to a further aspect of the present invention there is
provided a process for the preparation of compounds of formula (I)
wherein W represents the following groups as hereinbefore defined:
(4) (that is --SO.sub.2 NHR.sup.9), (5) (that is --SO.sub.3
R.sup.10) and (6) (that is --PO.sub.3 R.sup.10 R.sup.10) which
process comprises: reacting a compound of formula: ##STR10##
(wherein Z.sup.111 represents Z as hereinbefore defined with the
proviso that W represents the following groups as hereinbefore
defined: (4) (that is --SO.sub.2 NHR.sup.9), (5) (that is
--SO.sub.3 R.sup.10) and (6) (that is --PO.sub.3 R.sup.10
R.sup.10)); with compounds of formula (II) as hereinbefore defined
under standard conditions; or with compounds of formula (V) as
hereinbefore defined under standard conditions; whereby to form a
compound of formula I wherein W represents groups (4), (5) and (6),
and, if desired, converting the compound of formula I to a salt
thereof. Standard conditions include effecting the reaction with a
base (especially triethylamine) in the presence of an organic
solvent (preferably aprotic polar, especially dichloromethane),
preferably at ambient temperature. Compounds of formula (XVIII) are
known (available from Sigma Chemical Co.) or may be prepared from
known compounds by standard methods.
According to a further feature of the present invention there is
provided a process for the preparation of compounds of formula (Ia)
hereinbefore defined, wherein X is N or 0, Y is 0, R.sup.1 and
R.sup.2 are Cl, Br, I or OSO.sub.2 Me (especially Cl), R.sup.1a is
H, R.sup.2a is H, R.sup.3 is H and R.sup.4 is H, and the salts
thereof which process comprises reacting a compound of formula:
##STR11## (wherein R.sup.5a-d, Pr.sup.1 and Z.sup.1 are as
hereinbefore defined and X is 0 or NH) with either: a phosphorus
halogenating agent (especially phosphorus pentachloride) or thionyl
chloride in the presence of an organic solvent (preferably
non-polar aprotic, especially CH.sub.2 Cl.sub.2), preferably heated
at reflux for 1-2 h (especially 90 min); or methyl sulphonyl
chloride in the presence of an organic solvent (preferably polar
aprotic, especially pyridine) whereby to form a compound of Formula
(Ia) wherein X is 0 or NH, Y is 0, R.sup.1 and R.sup.2 are Br, I,
OSO.sub.2 Me or Cl, R.sup.1a is H, R.sup.2a is H, R.sup.3 is H and
R.sup.4 is H, and, if desired converting the compounds of formula
(Ia) into a salt thereof. When methyl sulphonyl chloride is used,
hydroxy groups in the compound of formula (XIX) maybe converted
into either Cl and/or OSO.sub.2 Me depending on the temperature
used for the reaction. The dichloro compounds (that is R.sup.1
.dbd.R.sup.2 .dbd.Cl) may conveniently be obtained by effecting the
reaction at 70.degree. C. for 15 min; and the corresponding
compound where R.sup.1 .dbd.Cl and R.sup.2 .dbd.OSO.sub.2 Me may
conveniently be obtained by effecting the reaction at 50.degree. C.
for 10 min.
When R.sup.1 and R.sup.2 are Br and R.sup.1 and R.sup.2 are I,
methane sulphonyl anhydride is preferably substituted for methane
sulphonyl chloride because this removes any problems of competing
halogen in the reaction. The compound of formula XIX (0.002M) was
dissolved in CHCl.sub.3 (30 ml). Triethylamine (1.12 ml) and
methysulphonyl anhydride (0.008M) were added at ambient
temperature, the mixture stirred for 2 hours and then washed with
water. The product so obtained was dried over MgSO.sub.4, filtered
and evaporated to an oil. The oil was dissolved in dry DMF and
lithium iodide (or bromide; 0.005M) added, stirred at 80.degree. C.
for 2 hours, cooled, poured into water and extracted with ether.
The product so obtained was dried over MgSO.sub.4, filtered,
evaporated to dryness and purified by flash column
chromatography.
Compounds of formula (XIX) may be prepared by reacting a compound
of formula: ##STR12## (wherein R.sup.5a-d are as hereinbefore
defined and R* respresents --N.dbd.C.dbd.O or --O--CO--L wherein L
represents a leaving group as hereinbefore defined) with a compound
of formula (III) as hereinbefore defined to obtain a compound of
formula: ##STR13## (wherein R.sup.5a-d, Pr.sup.1 and Z.sup.1 are as
hereinbefore defined and X is 0 or NH). The compound so obtained is
hydrogenated (preferably in the presence of palladium on carbon) to
obtain a compound corresponding to formula (XXI) but with
--NH.sub.2 in lieu of --NO.sub.2. The compound so obtained is
reacted with ethylene oxide under acid aqueous conditions
(prefrably acetic acid/water, 1:1), preferably for 1-2 days,
preferably at ambient temperature, whereby to form a compound of
formula (XIX). An example of the process is shown in Scheme 5.
A process for the preparation of compounds of formula (I), wherein
W represents a tetrazol-5-yl group, comprises reacting a compound
of formula: ##STR14## (wherein Z.sup.111 represents Z as
hereinbefore defined with the proviso that W represents a
tetrazol-5-yl group; with compounds of formula (II) as hereinbefore
defined under standard conditions; whereby to form a compound of
formula I wherein W represents a tetrazol-5-yl group and, if
desired, converting the compound of formula I to a salt thereof.
The standard conditions include reaction in a polar aprotic solvent
(especially DMF) in the presence of a base (preferably
dimethylamino pyridine, especially triethylamine) for at least 2 h
(preferably 20 h) at a temperature of 20.degree.-50.degree. C.
(especially 25.degree. C.).
In Vitro Cytotoxtc Potency of Prodrugs and Drugs
The in vitro cytotoxic potency of prodrugs, prodrugs plus enzyme
and drugs was measured in a cytotoxicity assay similar to that
described by Skehan et al (J. Natl. Cancer Inst 82, 1107-1112,
1990). LoVo cells (ECACC No: 87060101) were diluted in DMEM media
(containing 10% FCS, 1% glutamine and 0.2% gentamycin) plated out
in 96 well microtitre plates at a density of 2,500 cells/well and
incubated overnight at 37.degree. C. in 5% CO.sub.2. Various
concentrations of prodrug, corresponding drug as control or prodrug
plus enzyme (1U CPG2 activity/well--one unit of enzyme being the
amount required to hydrolyse 1 .mu.mole of methotrexate/min/ml at
37.degree.) were added to these cells and following either a 1 hr
or 24 hr incubation period the cells were washed, fresh medium
added and the cells incubated at 37.degree. C. in 5% CO.sub.2.
Three days after addition of compound, TCA was added to the wells
(16% final concentration) and the amount of cellular protein
adhering to the plates was assessed by the addition of SRB dye
(Skehan et al). The optical density at 540 nm was measured and
expressed as a percentage of the OD540 in control wells which
received no compound. The potency was expressed as the
concentration required to inhibit cell growth by 50% (IC.sub.50).
Prodrug on its own should generally possess low activity in the
test relative to prodrug in the presence of CPG2 (that is CPG2
enzyme is necessary for activation of prodrug to drug). Direct
addition of chemically synthesised drug (not needing CPG2
activation) acts as a control in the assay.
Using the methodology described above, representative compounds
tested of the invention were evaluated and were found to exhibit a
ratio of at least 10 fold greater activity in the presence of CPG2
compared with activity in the absence of CPG2 after 1 h; thereby
demonstrating and confirming the utility of the compounds of the
invention as effective.
According to a further aspect of the present invention we provide
pharmaceutical compositions comprising as pro-drug ingredient at
least one compound of Formula I or a physiologically acceptable
salt of said compound in association with a pharmaceutically
acceptable carrier or diluent. The composition will conveniently
comprise an effective amount of the pro-drug to be used with a CPG
(preferably CPG2)-tumour selective antibody conjugate already
localised at the tumour.
According to a further aspect of the present invention we provide
sterile pharmaceutical compositions for injection comprising as
pro-drug ingredient at least one compound of Formula I or a
physiologically acceptable salt of said compound in association
with a pharmaceutically acceptable carrier or diluent. The
composition will conveniently comprise an effective amount of the
pro-drug.
According to a further aspect of the present invention we provide a
two component system, each component for use in association with
the other, which comprises:
i) a first component that is an antibody or fragment thereof
capable of binding a given antigen, the antibody or fragment
thereof being conjugated to a CPG (preferably carboxypeptidase G2)
enzyme capable of converting a compound of formula I or
physiologically acceptable salt thereof into a cytotoxic drug;
and
ii) a second component that is a compound of formula I or a
physiologically acceptable salt thereof convertible under the
influence of the CPG (preferably carboxypeptidase G2) enzyme to the
cytotoxic drug.
The antibody or fragment thereof is preferably capable of binding
with a tumour associated antigen.
A particular antibody capable of binding with a tumour associated
antigen is mouse monoclonal antibody A5B7. Antibody A5B7 binds to
human carcinoembryonic antigen (CEA) and is particularly suitable
for targeting colorectal carcinoma. A5B7 is available from DAKO
Ltd., 16 Manor Courtyard, Hughenden Avenue, High Wycombe, Bucks
HP13 5RE, England, United Kingdom. Antibody fragments can be
prepared from whole IgG antibody by conventional means such as for
example F(ab').sub.2 fragments as described by Mariani, M. et al
(1991), Molecular Immunology 28, 69-77. In general the antibody (or
antibody fragment)--enzyme conjugate should be at least divalent,
that is to say capable of binding to at least 2 tumour associated
antigens (which may be the same or different). Antibody molecules
maybe humanised by known methods such as for example by "CDR
grafting" as disclosed in EP239400 or by grafting complete variable
regions onto human constant regions as disclosed in U.S. Pat. No.
4,816,567. Humanised antibodies may be useful for reducing
immunogenicity of an antibody (or antibody fragment). A humanised
version of antibody A5B7 has been disclosed in PCT WO92/01059.
The hybridoma which produces monoclonal antibody A5B7 was deposited
with the European Collection of Animal Cell Cultures, Division of
Biologics, PHLS Centre for Applied Microbiology and Research,
Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom. The date
of deposit was 14th Jul. 1993 and the accession number is No.
93071411. Antibody A5B7 may be obtained from the deposited
hybridoma using standard techniques known in the art such as
documented in Fenge C, Fraune E & Schuegerl K in "Production of
Biologicals from Animal Cells in Culture" (Spier R E, Griffiths J R
& Meignier B, eds) Butterworth-Heinemann, 1991, 262-265 and
Anderson B L & Gruenberg M L in "Commercial Production of
Monoclonal Antibodies" (Seaver S, ed), Marcel Dekker, 1987,
175-195. The cells may require re-cloning from time to time by
limiting dilution in order to maintain good levels of antibody
production.
Further antibodies useful in ADEPT have been described as follows.
Antibody BW 431/26 was described in Haisma, H. J. et al., Cancer
Immunol. Immunother., 34: 343-348 (1992). Antibodies-L6, 96.5, and
1F5 were described in European Patent 302 473. Antibody 16.88 was
described in International Patent Application WO90/07929. Antibody
B72.3 was described in European Patent No. 392 745. Antibody CEM231
was described in European Patent No. 382 411. Antibodies HMFG-1 and
HMFG-11 (Unipath Ltd, Basingstoke, Hants, United Kingdom) react
with a mucin-like glycoprotein molecule on milk fat globule
membranes and may be used to target breast and ovarian cancers.
Antibody SM3 (Chemicon International Ltd, London, United Kingdom)
reacts with core protein of mucin and may be used to target breast
and ovarian cancer. Antibodies 85A12 (Unipath Ltd, Basingstoke,
Hants, United Kingdom) and ZCEA1 (Pierce Chemical Company, Chester,
United Kingdom) react with tumour antigen CEA. Antibody PR4D1
(Serotec, Oxford, United Kingdom) reacts with a colon tumour
associated antigen. Antibody E29 (Dako Ltd, High Wycombe, United
Kingdom) reacts with epithelial membrane antigen. Antibody C242 is
available from CANAG Diagnostics, Gothenberg, Sweden.
Generally, antibodies useful in ADEPT are poorly internalised by
the tumour cells they recognise. This allows the targeted
prodrug-activating enzyme to be resident on the cell surface and
thus generate active drug at the tumour site from circulating
prodrug. Internalisation of antibody my be assayed by known
techniques, for example as set out in Jafrezou et al., Cancer
Research 52: 1352 (1992) and in Press et al., Cancer Research, 48:
2249 (1988).
Large scale purification of CPG2 from Pseudomonas RS-16 was
described in Sherwood et al (1985), Eur, J. Biochem., 148, 447-453.
Preparation of F(ab.sup.1).sub.2 and IgG antibodies coupled to CPG
enzyme may be effected by known means and has been described for
example in PCT WO 89/10140. CPG may be obtained from Centre for
Applied Microbiology and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, United Kingdom. CPG2 may also be obtained by
recombinant techniques. The nucleotide coding sequence for CPG2 has
been published by Minton, N. P. et al., Gene, 31 (1984), 31-38.
Expression of the coding sequence has been reported in E.coli
(Chambers, S. P. et al., Appl. Microbiol, Biotechnol. (1988), 29,
572-578) and in Saccharomyces cerevisiae (Clarke, L. E. et al., J.
Gen Microbiol, (1985) 131, 897-904). Total gene synthesis has been
described by H. Edwards in Am. Biotech. Lab (1987), 5, 38-44.
Expression of heterologous proteins in E. coli has been reviewed by
F. A. O. Marston in DNA Cloning Vol. III, Practical Approach
Series, IRL Press (Editor D H Glover), 1987, 59-88. Expression of
proteins in yeast has been reviewed in Methods in Enzymology Volume
194, Academic Press 1991, Edited by C. Guthrie and G R Fink.
CPG enzyme is available from Sigma Chemical Company, Fancy Road,
Poole, Dorset, U.K. CPG enzyme was described in: Goldman, P. and
Levy, C. C., PNAS USA, 58: 1299-1306 (1967) and in: Levy, C. C. and
Goldman P., J. Biol. Chem., 242: 2933-2938 (1967). Carboxypeptidase
G3 enzyme has been described in Yasuda, N. et al., Biosci. Biotech.
Biochem., 56: 1536-1540 (1992). Carboxypeptidase G2 enzyme has been
described in European Patent 121 352.
According to a further aspect of the present invention we provide a
method for the delivery of a cytotoxic drug to a site which
comprises administering to a host a first component, which first
component comprises an antibody or fragment thereof capable of
binding a given antigen, the antibody or fragment thereof being
conjugated to a CPG enzyme (preferably carboxypeptidase G2) capable
of converting a compound of formula I or physiologically acceptable
salt thereof into a cytotoxic drug; followed by administration to
the host of a second component, which second component comprises a
compound of formula I or a physiologically acceptable salt thereof
convertible under the influence of the CPG enzyme (preferably
carboxypeptidase G2) to the cytotoxic drug.
The site to which the cytotoxic drug is to be delivered is
preferably tumour cells which will generally be present in a
tumour-bearing mammalian host such as a human.
When the said first component is administered to the tumour bearing
host, the antibody or antibody fragment moiety of the conjugate
directs the conjugate to the site of the tumour and binds the
conjugate to the tumour cells.
Once unbound conjugate has been substantially eliminated from the
host to be treated, for example by clearance from the host after
the elapsing of an appropriate time or after accelerated clearance,
for example as described in Br. J. Cancer (1990), 61, 659-662, the
second component maybe administered to the host. It is highly
desirable to substantially eliminate unbound conjugate from the
host before administration of the second component, since otherwise
cytotoxic drug may be generated other than at the site of the
tumour thus resulting in general toxicity to the host rather than
site specific toxicity.
The compounds of this invention may be utilized in compositions
such as tablets, capsules or elixirs for oral administration,
suppositories for rectal administration, sterile solutions or
suspensions for parenteral or intramuscular administration, and the
like.
The compounds of this invention can be adminstered to patients
(animals and human) in need of such treatment in dosages that will
provide optimal pharmaceutical efficacy. Although the dose will
vary from patient to patient depending upon the nature and severity
of disease, the patient's weight, special diets then being followed
by a patient, concurrent medication, and other factors which those
skilled in the art will recognize, the dosage range will generally
be about 1 to 150 mg. per kg per patient per day which can be
administered in single or multiple doses. Preferably, the dosage
range will be about 10 to 75 mg. per kg per patient per day; more
preferably about 10 to 40 mg. per kg per patient per day.
Naturally, these dose ranges can be adjusted on a unit basis as
necessary to permit divided daily dosage and, as noted above, the
dose will vary depending on the nature and severity of the disease,
weight of patient, special diets and other factors.
Typically, these dosages can be formulated into pharmaceutical
compositions as discussed below.
About 50 to 500 mg. of compound or mixture of compounds of Formula
1 or a physiologically acceptable salt thereof is compounded with a
physiologically acceptable vehicle, carrier, excipient, binder,
preservative, stabilizer, flavor, etc., in a unit dosage form as
called for by accepted pharmaceutically practice. The amount of
active substance in these compositions or preparations is such that
a suitable dosage in the range indicated is obtained.
Illustrative of the adjuvants which can be incorporated in tablets,
capsules and the like are the following: a binder inch as gum
tragacanth, acacia, corn starch or gelatin; an excipient such as
microcrystalline cellulose; a disintegrating agent such as corn
starch, pregelatinized starch, alginic acid and the like; a
lubricant such as magnesium stearate; a sweetening agent such as
sucrose, lactose or saccharin; a flavoring agent such as
peppermint, oil of wintergreen or cherry. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier such as fatty oil. Various other materials
may be present as coatings or to otherwise modify the physical form
of the dosage unit. For instance, tablets may be coated with
shellac, sugar or both. A syrup or elixir may contain the active
compound, sucrose as a sweetening agent, methyl and propyl parabens
as preservatives, a dye and a flavouring such as cherry or orange
flavour.
As discussed above the compounds of formula I and the
physiologically acceptable salts thereof as well as the conjugates
referred to above maybe administered using conventional modes of
administration including, but not limited to intravenous,
intraperitoneal, oral, intralymphatic or adminstration directly
into the tumour. Intravenous administration is preferred, for
example intravenous infusion.
Sterile compositions for injection or infusion can be formulated
according to conventional pharmaceutical practice by dissolving or
suspending the active substance in a vehicle such as water for
injection, a naturally occurring vegetable oil like sesame oil,
coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fatty
vehicle like ethyl oleate or the like. Buffers, preservatives,
antioxidants and the like can be incorporated as required.
Preferred sterile compositions for injection or infusion formulated
according to conventional pharmaceutical practice include
dissolving the prodrug in a vehicle such as water optionally
containing salts, sugar (especially dextran), buffering agents
and/or co-solvents (especially polyethylene glycol, propylene
glycol or dimethyl isosorbide).
In one embodiment of the present invention, the compound of formula
I is provided in the form of the free acid which free acid may then
be formulated into a form for parenteral administration immediately
prior to administration to the patient. Thus, for example, the
compound of formula I in the form of the free acid may be mixed
with a buffer whereby it is converted to a physiologically
acceptable salt immediately prior to administration.
A preferred unit dosage form of a compound of the present invention
comprises a compound of formula I, or a pharmaceutically acceptable
salt thereof, in sterile freeze-dried form for reconstitution into
injectable/infusable solution in an ampoule. The ampoule preferably
contains 500 mg to 2 g (especially 1 g) of said compound.
The following examples illustrate the preparation of the compounds
of formula I and their incorporation into pharmaceutical
compositions and as such are not to be considered as limiting the
invention. In the examples, unless otherwise stated, the following
items were standard procedures:
i) All evaporations were carried out by rotary evaporation in vacuo
and work-up procedures were carried out after removal of residual
solids by filtration;
ii) operations were carried out at room temperature, that is in the
range 18.degree.-25.degree. C. and under an atmosphere of an inert
gas such as argon;
iii) column chromatography (by the flash procedure) was performed
on Merck Kieselgel silica (Art. 9385) obtained from E. Merck,
Darmstadt, W. Germany;
iv) yields are given for illustration only and are not necessarily
the maximum attainable;
v) the end-products of the formula I have satisfactory
microanalyses and their structures were confirmed by NMR and mass
spectral techniques;
vi) intermediates were not generally fully characterised and purity
was assessed by thin layer chromatographic, infra-red (IR) or NMR
analysis;
vii) melting points are uncorrected and were determined using an
oil-bath apparatus; melting points for the end-products of the
formula I were determined after crystallisation from a conventional
organic solvent such as ethanol, methanol, acetone, ether or
hexane, alone or in admixture.
EXAMPLE 1
N-(4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl)-L-glutamic
acid
A solution of dibenzyl
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamate (see
(2) in scheme 1) (6 g) in ethyl acetate (100 ml) was hydrogenated
over 30% palladium on carbon (0.6 g) for 2 h. When the theoretical
amount of hydrogen had been taken up the catalyst was removed by
filtration and the filtrate evaporated to dryness. The residue was
taken up into hot ether (25 ml) and hexane added until cloudy. On
cooling
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic acid
(see. (3) in Scheme 1) was obtained as a white crystalline solid
(3.4 g) 79% yield m.p. 87.degree.-89.degree..
NMR 7.0 (d) 2H; 6.6 (d) 2H; 6.2 (d) 1H; 4.4 (m) 1H; 3.5-3.7 (m) 8H;
2.0-2.6 (m) 4H.
Elemental Analysis--Expected C=47.2, H=4.95, N=6.88 Found C=47.5,
H=5.1, N=6.7.
The titled compound was also prepared in a different polymorphic
form, not soluble in ether, with a melting point of
128.degree.-130.degree. C.
The starting material dibenzyl
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamate was
prepared as described below.
A solution of 4-nitrophenyl-chloroformate (1.43 g) in chloroform
(15 ml) was added to a mixture of 4-[bis(2-chloroethyl)amino]phenol
hydrochloride (Biochem. Pharmacol 17 893 (1968)) (1.93 g),
triethylamine (2 ml) and chloroform (20 ml). After 2 h at ambient
temperature the mixture was evaporated to dryness and the residue
chromotographed on Merck silica gel Art 9385. On elution with
hexane/ethyl acetate and recrystallisation from benzene:petroleum
ether (3:1) the product
0-(4-[N,N-bis(2-chloroethyl)amino]phenyl)-0'-(4-nitrophenyl)carbonate
(see (1) in Scheme 1) was obtained as a yellowish solid (1.4 g)
(50%) mp=66.degree.-7.degree..
Triethylamine (3.8 ml) was added to 5.5 g of the product so
obtained in chloroform (40 ml), followed by addition of L-Glutamic
acid dibenzyl ester tosylate (13.75 g). The mixture was stirred and
heated at 60.degree. for 4 h and evaporated to dryness. The residue
was chromatographed on silica gel (Merck Art 9385) and eluted with
3% ethyl acetate in chloroform to obtain the required starting
material
dibenzyl-2-([bis(2-chloroethyl)amino]phenoxycarbonyl)glutamate (see
(2) in Scheme 1) 6.2 g (77% yield) as a white solid m.p.
85.degree.-7.degree..
EXAMPLE 2
N-(4-[N,N-bis(2-chloroethyl)-amino]-3-methylphenoxycarbonyl)-L-glutamic
acid
The process described in Example 1 was repeated using dibenzyl
N-(4-[N,N-bis(2-chloroethyl)amino]-3-methyl-phenoxycarbonyl)-L-glutamate
in place of dibenzyl
N-(4-[N,N-bis(2-chloroethyl)-amino]phenoxycarbonyl)-L-glutamate to
obtain
N-(4-[N,N-bis(2-chloroethyl)amino]-3-methyl-phenoxycarbonyl)-L-glutamic
acid as a white solid, m.p. 160.degree.-162.degree. C.
NMR: 7.3 (d) 2H; 6.7 (d) 2H; 6.0 (d) 1H, 4.2 (m) 1H, 3.7 (m) 8H;
2.3-2.0 (m) 4H.
Elemental Analysis: Expected C=47.3; H=5.2; N=10.3; Cl=17.5 Found
C=46.8; H=5.2; N=10.3; Cl=18.0.
The dibenzyl
N-(-4-[N,N-(2-chloroethyl)amino]-3-methylphenoxycarbonyl)-L-glutamate
used as starting material was prepared in an analogous manner to
that described in Example 1 using
4-[N,N-bis(2-chloroethyl)amino]-3-methyl-phenol in place of
4-[N,N-bis(2-chloroethyl)amino]phenol.
The 4-[N,N-bis(2-chloroethyl)amino]-3-methyl-phenol was obtained as
follows (see Scheme 2).
(a) Ethylene oxide (40 g) was bubbled into a solution of
4-amino-m-cresol (12.3 g) in acetic acid/water (1:1) (500 ml). The
mixture was allowed to remain at room temperature for 48 h and then
evaporated to dryness. The residue was chromatographed on silica
gel, eluted with ethyl acetate to obtain
4-[N,N-bis(2-hydroxyethyl)amino]-3-methylphenol as an oil (6.2
g).
NMR: See Table 2.
(b) Benzyl bromide (4.24 g) was added to a mixture of the product
so obtained (6 g), potassium hydroxide (1.6 g) and ethanol (40 ml).
The mixture was stirred and heated at reflux for 2 h, cooled and
concentrated by evaporation. The residue was poured onto water (100
ml), extracted twice with ethyl acetate, dried and evaporated to
obtain 2,2'-(4-benzyloxy-2-toluidino)diethanol as a solid (7.2 g)
mp=70.degree.-72.degree. C.
NMRTable 3
(c) Phosphorous pentachloride (11.4 g) was added in portions to the
product so obtained (7 g) in chloroform (50 ml) at
10.degree.-20.degree. C. The mixture was then heated at reflux for
90 min, cooled and poured onto water. The organic phase was
separated and washed with aqueous sodium bicarbonate solution,
water and evaporated to dryness. The residue was chromatographed on
silica gel. After elution with hexane/ethyl acetate (2:1)
4-benzyloxy-3-methyl-N,N-bis(2-chloroethyl)-aniline (2.2 g) was
obtained as an oil.
NMR: See Table 4
An alternative reaction for use in lieu of the reaction immediately
above is as follows. Methane sulphonyl chloride (2.5 ml) was added
at 0.degree.-5.degree. C. to a solution of the product obtained in
step (b) (2.6 g) in pyridine (8 ml). The mixture was then heated at
70.degree. C. for 15 min, cooled and poured onto dilute citric acid
solution (100 ml). The mixture was extracted twice with ethyl
acetate, dried and evaporated to obtain
4-benzyloxy-3-methyl-N,N-bis(2-chloroethyl)aniline as an oil 2.6 g
(84%).
NMR: Table 4.
(d) Ethereal HCl (saturated) was added to the product so obtained
(2 g) in ethanol (25 ml) until complete solution was observed. 300
mg of 30% palladium on carbon catalyst was added and the mixture
stirred under an atmosphere of hydrogen until the appropriate
amount of hydrogen was taken up. The catalyst was removed by
filtration and the filtrate evaporated to obtain
4-[N,N-bis(2-chloroethyl)]amino-3-methylphenol hydrochloride as a
solid (950 mg) mp=164-7.
EXAMPLE 3
N-(-4-[N,N-bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid
hydrochloride
A saturated solution of hydrogen chloride in ether (120 ml) was
added to a solution of ditertbutyl
N-(-4-[N,N-bis-(2-chloroethyl)amino]-phenylcarbamoyl)-L-glutamate
(4.4 g) in ethyl acetate (20 ml). After 1 hour at ambient
temperature the mixture was evaporated to a solid. This solid was
triturated with ether to obtain
N-(4-[N,N-bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid
hydrochloride (3.5 g) as a grey solid m.p=148.degree.-150.degree.
C. (see Scheme 3)
NMR: 7.2 (d) 2H; 6.7 (d) 2H); 4.2 (n) 1H; 3.7 (m) 8H; 2.4-1.8 (m)
4H;
Elemental Analysis: Found C=46.7; H=6.8; N=7.0 Expected C=47.1;
H=6.5; N=7.5.
The starting material ditertbutyl
N-(4-[N,N-bis-[(2-chloroethyl)amino]-phenylcarbamoyl)-L-glutamate
was obtained as follows:
A mixture of p-fluoronitrobenzene (14.1 g) and diethanolamine (30
ml) was stirred and heated at 130.degree. C. for 2 h. The mixture
was cooled to about 60.degree. C. and poured onto 1 L of water
containing 10 ml of 48% caustic soda solution. After cooling to
15.degree. C. a precipitate was filtered off and dried to obtain
2,2'-(4-nitroanilino)diethanol (20.7 g) (92%)
m.p.=102.degree.-104.degree. C.
Thionyl chloride (30 ml) was added, with cooling, to a mixture of
the product so obtained (20 g), dichloromethane (200 ml) and
pyridine (7 ml). After the addition the mixture gas heated at
reflux for 1 hour. After cooling the mixture was diluted with an
equal volume of dichloromethane and carefully washed twice with
water, dried and evaporated to obtain
[N,N-bis(2-chloroethyl)]-4-nitro-aniline as a solid 21 g
m.p.=81.degree.-3.degree. C.
To a solution of the product so obtained (0.53 g) in redistilled
tetrahydrofuran (20 ml) was added 30% palladium on carbon catalyst
(100 mg). The mixture was stirred under an atmosphere of hydrogen
for 2 h and the catalyst then removed by filtration. The filtrate
was evaporated to dryness and the residue redissolved in ether (20
ml) and a solution of hydrogen chloride gas in ether added to
slight excess. The resulting
4-[N,N-bis(2-chloroethyl)amino]anilinium chloride was obtained as a
solid and dried. Yield=0.5 g m.p. 238.degree.-40.degree. C.
(d).
To a solution of triphosgene (Aldrich) 200 mg in chloroform (10 ml)
at 0.degree.-5.degree. C., was added the product so obtained (539
mg) followed by triethylamine (0.83 ml). After 15 min at room
temperature a solution of L-glutamic acid ditertbutyl ester (0.31
g) in chloroform (5 ml) was added. The mixture was allowed to stand
at ambient temperature for 18 h, washed with water, dried and
evaporated to dryness. The residue was chromatographed on Merck
silica gel and eluted with hexane--ethyl acetate (3:1) to obtain
the desired starting material ditertbutyl
4[bis-2-(chloroethylamino)]phenylcarbamoyl-L-glutamate 0.44 g as an
oil (see Scheme 3).
NMR: .delta.7.2d and .delta.6.65 (dd)4H. aromatics .delta.4.1(m)1H
.delta.3.66(S)8H .delta.1.7-2.2(m)4H .delta.1.38(s)9H and
.delta.1.42(s)9H
EXAMPLE 4
N-(-4-[N,N-bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic
acid
An alternative process to that described in Example 3 for
preparation of
N-(4-[N,N-bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid
is described below, The starting material dibenzyl
N-(4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamate was
prepared in an analogous manner to the corresponding step in
Example 5.
A solution of dibenzyl N-(4-[N,N-bis-(2-chloroethyl)
amino]-phenylcarbamoyl)-L-glutamate (1.138 g) in DMF (15 ml) was
hydrogenated over 10% Pd/C for 16 h. After filtration and
evaporation in vacuo, the residue was dissolved in CHCl.sub.3 (20
ml). After 18 h the crystalline precipitate was filtered off and
dried in vacuo to obtain
N-(4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic
acid. Yield, 730 mg (93%). After recrystallization from
acetone/CHCl.sub.3 microscopic rods formed m.p.
116.degree.-118.degree..
NMR (CD.sub.3 COCD.sub.3): .delta. 8.0(s)1H; 7.2(d)2H; 6.6(d)2H;
6.2(d)2H NH; 4.4(m)1H; 3.6(m)8H; 2.5-1.9(m)4H.
EXAMPLE 5
N-(4-[N,N-bis(2-chloroethyl)-amino]-3-fluorophenylcarbamoyl)-L-glutamic
acid
To a solution of dibenzyl
N-(4-[N,N-bis(2-chloroethyl)-amine]-3-fluorophenyl-carbamoyl)
L-glutamate (0.4 g) in ethyl acetate (10 ml) was added 30%
palladium on carbon (50% moist) (160 mg) and the mixture stirred
under an atmosphere of hydrogen for 1 hour. After filtration of the
catalyst the filtrate was evaporated to dryness.
N-(4-[N,N-bis(2-chloroethyl)-amine]3-fluorophenylcarbamoyl
L-glutamic acid was obtained after trituration of the oily residue
with ethyl acetate/hexane as a white powder (210 mg, mp
111.degree.-114.degree. C.). The starting material dibenzyl
N-(4-[N,N-bis(2-chloroethyl)amino]-3-fluorophenylcarbamoyl)L-glutamic
acid was obtained as follows:
A suspension of 4-[N,N-bis-(2-chloroethyl)amino]3-fluoroanilinium
oxalate (3.5 g) in anhydrous ethyl acetate (200 ml) and potassium
carbonate (5.5 g) was cooled under argon to 5.degree. C. To this
mixture was added a solution of phosgene in toluene (1.9M; 5.5 ml).
After the addition the mixture was stirred at room temperature for
10 min, filtered and the filtrate dried over magnesium sulphate.
The dried filtrate obtained was added in one portion to a mixture
of dibenzyl glutamate p toluenesulphonate (5 g), potassium
carbonate (2 g) and ethyl acetate (100 ml). Triethylamine (2 ml)
was added and the mixture stirred for 20 min at ambient
temperature. The mixture was filtered and the filtrate evaporated
to dryness. The residue was chromatographed on silica gel eluting
with ethyl acetate/hexane (1:2) to obtain the desired starting
material as an oil, which crystallized. Yield=5.5 g m.p.
81.degree.-84.degree. C.
4-[N,N-bis(2-chloromethyl)amino]-3-fluoroanilium oxalate was
prepared as described in Example 3 except that 3,4
difluorotrobenzene was used as starting material in place of
p-fluoronitrobenzene to obtain
4-[N,N-bis-(2hydroxyethyl)amino]fluoronitrobenzene
m.p.=99.degree.-101.degree. C.
The produce so obtained was treated with thionyl chloride as
described in Example 3 to obtain
4[N,N-bis(2-chloroethyl)-3-fluoronitrobenzene. m.p. 66.degree.14
8.degree. C.
The product so obtained was hydrogenated as described in Example 3
using ethyl acetate as solvent. The mixture was filtered and the
filtrate evaporated to low volume and redissolved in ether. A
saturated solution of oxalic acid in ether was added to excess and
the desired product
4-[N,N-bis(2-chloromethyl)amino]-3-fluoroanilinium oxalate
collected. m.p=146.degree.-8.degree. C.
EXAMPLE 6
N-(4-[N,N-bis(2-chloroethyl)amino]-3-chlorophenylcarbamoyl)-L-glutamic
acid
A solution of dibenzyl
N-((4[N,N-bis-(2-chloroethyl)amino]3-chlorophenylcarbamoyl))-L-glutamate
(350 mg) in ethyl acetate (30 ml) containing 30% palladium on
carbon 70 mg (50% moist) was stirred under an atmosphere of
hydrogen for 1 hour, After filtration of the catalyst the filtrate
was evaporated to dryness and the residue triturated with
ether/ethyl acetate to obtain N-(4-[N,N-bis
(2-chloroethyl)amino]-3-chlorophenylcarbamoyl)-L glutamic acid as
an oil.
NMR: 8.7 (s) 1H; 7.6 (s) 1H; 7.1-7.4 (m) 2H; 6.5 (d) 1H; 4.2 (m)
1H; 3.3-3.6 (m) 8H, 1.7-2.4 (m) 4H.
The starting material dibenzyl 4-[N,N-bis-(2-chloroethyl)amino
3-chlorocarbamoyl)-L-glutamate was obtained by a procedure similar
to Example 3 except that 4-fluoro-3-chloronitrobenzene was used as
starting material in place of 4-fluoronitrobenzene to obtain
4[N,N-bis-(2-hydroxyethylamino)]-3-chloro-nitrobenzene as an orange
oil.
NMR 8.0-8.2 (m) 2H; 7.3 (l) 1H; 4.7 (t) 2H, 3.5(m) 8H.
The product so obtained was treated with thionyl chloride as
described in Example 3 to obtain 4[N,N-bis-2
chloroethylamino)-3-chloronitrobenzene as an oil.
NMR (CHCl.sub.3) 8.3 (d) 1H; 8.1 (q) 1H; 7.2 (d) 1H; 3.8 (t) 4H;
3.6 (t) 4H.
The product so obtained was hydrogenated using ethyl acetate as
solvent as described in Example 3. The catalyst was removed by
filtration and the filtrate evaporated to low volume, redissolved
in ether and saturated ethereal oxalic acid added to excess. The
oxalate salt was obtained by filtration m.p.=118.degree.-21.degree.
C.
The product so obtained was converted to dibenzyl
N-(4[N,N-bis-(2-chloroethyl)amino]-3-chlorophenylcarbamoyl)-L-glutamic
acid as described in Example 5. An oil was obtained;
NMR 7.1-7.4 (m) 13H; 5.1 (s) 2H; 5.0 (s) 2H; 4.6 (m) 1H; 3.5 (s)
8H; 2.0-2.6 (m) 4H.
EXAMPLE 7
Typical Pharmaceutical Compositions Containing a Compound of The
Invention
A: Dry Filled Capsules Containing 100 mg of Prodrug Per Capsule
______________________________________ Amount per Ingredient
capsule (mg) ______________________________________ Compound 100
Lactose 149 Magnesium stearate 1 Capsule (size No 1) 250
______________________________________
The compound can be reduced to a No. 60 powder and the lactose, and
magnesium stearate can then be passed through a No. 60 blotting
cloth onto the powder. The combined ingredients can then be mixed
for about 10 minutes and filled into a No. 1 dry gelatin
capsule.
B: Tablet
A typical tablet would contain compound (100 mg), pregelatinized
starch USP (82 mg), microcrystalline cellulose (82 mg) and
magnesium stearate (1 mg).
C: Suppository
Typical suppository formulations for rectal administration can
contain compound (50 mg), disodium calcium edetate (0.25-0.5 mg).
and polyethylene glycol (775-1600 mg). Other suppository
formulations can be made by substituting, for example butylated
hydroxytoluene (0.04-0.08 mg) for the disodium calcium edetate and
a hydrogenated vegetable oil (675-1400 mg) such as Suppocire L,
Wecobee F S, Wecobee M, Witepsols, and the like, for the
polyethylene glycol.
D: Injection
A typical injectible formulation would contain compound (500 mg)
benzylalcohol (0.05 ml) and 0.15M sodium bicarbonate for injection
(5.0 ml).
EXAMPLE 8 (see Scheme 4)
N-(4-[N-(2-chloroethyl)-N-(2-mesyloxyethyl)amino]phenoxycarbonyl)-L-glutami
c acid
N-(4-([N-(2-chloroethyl),
N-(2-mesyloxyethyl)]amino)-phenyloxycarbonyl)-L-glutamic
acid-di-t-butyl ester (6, wherein R.sup.1 -Cl and R.sup.2
=OSO.sub.2 Me) (110 mg) was suspended in trifluoroacetic acid (TFA)
(2.2 ml) and stirred for 40 min at ambient temperature. TFA was
removed under reduced pressure; the remaining oil was diluted with
ethyl acetate (1 ml) and evaporated to obtain
N-(4-[N-(2-chloroethyl)-N-(2-mesyloxyethyl)amino]phenoxycarbonyl)-L-glutam
ic acid -1.02 TFA -0.16 EtOAc (9, wherein R.sup.1 =Cl and R.sup.2
--OSO.sub.2 Me), 90 mg, 95% yield.
NMR: --CH.sub.2 --CH.sub.2 --OSO.sub.2 Me/--CH.sub.2 --CH.sub.2 Cl
3.15 (s), 3H; 3.70 (m) 6H; Other 4.30 (t) 1H; 4.49 (t) 1H;
Aromatics 6.75 (d) 2H; 6.92 (d) 2H; 1.8-2.3 (m) 4H; 4.02 (m) 1H;
7.90 (d) 1H.
The starting material
4-([N-(2-chloroethyl),N-(2-mesyloxyethyl)]amino)phenyloxycarbonyl-L-glutam
ic acid-di-t-butyl ester was prepared as described below.
A solution of L-glutamic acid di-t-butyl ester hydrochloride (4.26
g) and triethylamine (4 ml) in dry chloroform (30 ml) was stirred
with a cooled solution of 4-nitrophenylchloroformate (2.92 g,
available from Aldrich) for 5 min. After 5 hours at ambient
temperature, the solvent was evaporated and the residue dissolved
in ethyl acetate (70 ml), filtered and evaporated to dryness. The
residue was chromatographed on silica gel; eluted with chloroform
to obtain 4-nitrophenyloxycarbonyl-L-glutamic acid di-t-butyl ester
(2) as an oil, 5.02 g (82%).
NMR: 7.33 (d) 2H; 8.24 (d) 2H; 1.46 (s) 9H; 1.50 (s) 9H; 2.0-2.4
(M) 4H; 4.32 (m) 1H; 5.90 (d) 1H.
A solution of the product so obtained (2) (5.01 g) in acetic acid
(30 ml) was hydrogenated over 10% palladium on carbon for three
days. After filtering, the solution was cooled and ethylene oxide
(5 ml) added, and left at ambient temperature for 22 hours. Solvent
was evaporated and the residue was partitioned between ethyl aceate
and water. The organic phase was separated, washed with water,
dried (Na.sub.2 SO.sub.4) and evaporated to dryness. The residue
was chromatographed on silica gel; eluted with ethyl acetate in
chloroform (2:1) to obtain
4-[bis(2-hydroxyethyl)amino]phenyloxycarbonyl-L-glutamic acid
di-t-butyl ester (4) (3.93 g) 69% m.p. 91.degree.-93.degree. C.
A solution of the product so obtained (4) (0.86 g) in pyridine (3
ml) was stirred with methanesulphonyl chloride (0.6 ml) at
2.degree. C. for 20 min, followed by 50.degree. C. for 10 min. The
reaction mixture was partitioned between ethyl acetate and water.
The organic phase was separated, washed with water, dried (Na.sub.2
SO.sub.4) and evaporated to dryness. The residue was
chromatographed on silica gel; eluted with ethyl acetate in
dichloromethane (1:9) to obtain
4-[(2-chloroethyl)[2-(mesyloxy)ethyl]aminophenyloxycarbonyl-L-glutamic
acid-di-t-butyl ester (6) as an oil (0.44 g) 43%.
NMR: 3.15 (s) 3H; 3.70 (m) 6H; 4.29 (t) 2H; 6.75 (d) 2H; 6.92 (d)
2H; 1.41 (s) 9H; 1.42 (s) 9H; 1.8-2.3 (m) 2H; 3.97 (m), 1H; 7.92
(d) 1H.
EXAMPLES 9-15
The following compounds listed in Table 1 were prepared according
to Example 2 using the starting materials and intermediates listed
in Tables 2-8 below:
TABLE 1
__________________________________________________________________________
##STR15## Ex No R.sup.5a-b mpt ClCH.sub.2 CH.sub.2 M Aromatics
.alpha.CH CH.sub.2 CH.sub.2 Other
__________________________________________________________________________
9 CH.sub.3 (R.sup.5a) 160-2.degree. C. 3.34-3.54(m)8H
6.9-7.22(m)3H; 4.1(m)1H 2.0-2.5(m)4H 2.27(s)3H CH.sub.3 10 Pr.sup.i
(R.sup.5a) 156-8.degree. C. 3.32-3.55(m)8H 6.95-7.28(m)3H; 4.1(m)1H
2.0-2.49(m)4H 3.7(m)1H CH 1.14(d)2H(CH.sub.3). sub.2 11 CH.sub.3
(R.sup.5b) 124-6.degree. C. 3.7(m)8H 6.5-6.9(m)3H 4.1(m)1H
1.9-2.3(m)4H 2.1(s)3H CH.sub.3 12 F(R.sup.5a) 3.6(m)8H 6.8-7.2(m)3H
4.1(m)1H 2.0-2.49(m)4H 13 CHCHCHCH 3.6(m)8H 6.6-8.0(m)6H 4.2(m)1H
2.1-2.5(m)4H (R.sup.5a-b) 14 Cl(R.sup.5b) 106-8.degree. C. 3.6(m)8H
6.7-7.1(m)3H 4.1(m)1H 1.9-2.4(m)4H 15 Cl(R.sup.5a) 148-150.degree.
C. 3.4-3.54(m)8H 7.0-7.4(m)3H) 4.1(m)1H 1.9-2.36(m)4H
__________________________________________________________________________
Thus the compounds of Examples 9-15 were prepared in an analogous
manner to that described in Example 2. The compound of Example 10
was thus prepared by substituting 4-amino-3-isopropylphenol
(Gilman, H, et al., J. Org. Chem. 19, (1954) 1067-78 for
4-amino-m-cresol used in step (a). The compound of Example 11 was
prepared by substituting 4-amino-2-methylphenol (available from
Aldrich) for 4-amino-m-cresol and the compound of Example 12 was
prepared by substituting 4-amino-3-fluorophenol (prepared according
to Journal of the Chemical Society (1964) p473) for
4-amino-m-cresol. The compound of Example 13 was prepared by
substituting 4-aminonaphth-1-ol (Aldrich Chemical Co Ltd) for
4-amino-m-cresol and the compound of Example 14 was prepared by
substituting 4-amino-2-chlorophenol (prepared according to Journal
of the American Chemical Society 45, 2192, (1923)) for
4-amino-m-cresol. The compound of Example 15 was prepared by
substituting 4-amino-3-chloro-phenol (Berichte; p. 2065 (1938); and
Organic Synthesis; Collected Vol 4, p. 148) for
4-amino-m-cresol.
The starting materials and intermediates employed in the
preparation of Examples 9-15 and their properties are listed in
Tables 2-8 below:
TABLE 2
__________________________________________________________________________
##STR16## R5.sup.a or .sup.b m.p. or NMR data Reference
__________________________________________________________________________
Me (R5.sup.a) 176-9.degree. C. Aldrich Pr.sup.i (R5.sup.a)
172-5.degree. C. Cl (R5.sub.a) 6.5-6.7(m)3H Aromatics; J. Chem.
Soc. 8.8(s)1H OH; (1928), 2703 4.6(br s)2H NH.sub.2 ; 159.5.degree.
C. Me (R5.sup.b) 174-6.degree. C. Aldrich Cl (R5.sub.b)
146-8.degree. C. F (R5.sub.a) 6.6-6.9(m)3H Aromatics; 9.4(s)1H OH;
4.4(m)2H NH.sub.2 DiCH.sub.3 (R5.sup.a = R5.sup.b = 260-2.degree.
C. (as hydrochloride ex Aldrich) CH.sub.3) (R5.sup.a and R5.sup.b
273.degree. C. (as hydrochoride ex Aldrich) together represent
CHCHCHCH)
__________________________________________________________________________
TABLE 3
__________________________________________________________________________
##STR17## R5.sup.a-b HOCH.sub.2 CH.sub.2 N Aromatics Other
__________________________________________________________________________
Me (R5.sup.a) 2.8-3.3(m)8H 6.4-6.9(m)3H 2.03(s)3H CH.sub.3 Pr.sup.i
(R5.sup.a) 3.0-3.4(m)8H 6.6-7.1(m)3H 3.6(m)1H; 1.1(d)6H Pr.sup.i Cl
(R5.sup.a) 3.15-3.54(m)8H 6.7-7.2(m)3H CH.sub.3 (R5.sup.b)
3.3-3.5(m)8H 6.4-6.6(m)3H 2.06(s)3H CH.sub.3 Cl (R5.sup.b)
3.2-3.6(m)8H 6.5-6.8(m)3H F (R5.sup.a) 3.1-3.4(m)8H 6.5-6.9(m)3H di
CH.sub.3 2.9-3.4(m)8H 6.6-7.0(m)3H 2.0(s)3H; 2.2(s)3H CH.sub.3
(R5.sup.a = R5.sup.b = CH.sub.3) (R5.sup.a and R5.sup.b
3.2-3.4(m)8H 6.8-8.3(m)11H together represent CHCHCHCH
__________________________________________________________________________
TABLE 4
__________________________________________________________________________
##STR18## R5.sup.a-b HOCH.sub.2 CH.sub.2 N Aromatics CH.sub.2 Other
__________________________________________________________________________
Me (R5.sup.a) 3.0-3.2(m)8H 6.8-7.5(m)8H 5.0(s)2H 2.2(s)3H; CH.sub.3
Pr.sup.i (R5.sup.a) 2.9-3.3(m)8H 6.8-7.5(m)8H 5.0(s)2H 3.6(m)1H;
1.1(d)6H Pr.sup.i Cl (R5.sup.a) 3.05-3.35(m)8H 6.9-7.5(m)8H
5.1(s)2H 2.2(s)3H CH.sub.3 CH.sub.3 (R5.sup.b) 3.1-3.4(m)8H
6.9-7.3(m)8H 5.0(s)2H Cl (R5.sup.b) 3.3-3.5(m)8H 6 8-7.4(m)8H
5.0(s)2H F (R5.sup.a) 3.2-3.4(m)8H 6.8-7.5(m)8H 5.0(s)2H Di
CH.sub.3 2.9-3.3(m)8H 6.8-7.4(m)8H 5.0(s)2H 2.2(s)3H; 2.1(s)3H
CH.sub.3 (R5.sup.a = R5.sup.b = CH.sub.3) (R5.sup.a and R5.sup.b
3.2-3.4(m)8H 6.9-8.3(m)6H 5.3(s)2H together represent CHCHCHCH)
__________________________________________________________________________
TABLE 5
__________________________________________________________________________
##STR19## R5.sup.a-b R.sup.1 R.sup.2 CH.sub.2 CH.sub.2 N CH.sub.2
Aromatics Other
__________________________________________________________________________
Me(R5.sup.a) Cl Cl 3.3-3.5(m)8H 5.08(s)2H 6.8-7.4(m)8H 2.3(s)3H Me
Pr.sup.i (R5.sup.a) Cl Cl 3.2-3.5(m)8H 5.04(s)2H 6.8-7.4(m)8H
3.7(m)1H; 1.1(d)6H Cl(R5.sup.a) Cl Cl 3.4-3.6(m)8H 5.1(s)2H
6.9-7.4(m)8H CH.sub.3 (R5.sup.b) Cl Cl 3.3-3.6(m)8H 5.1(s)2H
6.9-7.4(m)8H Cl(R5.sup.b) Cl Cl 3.6(m)8H 5.1(s)2H 6.8-7.4(m)8H
F(R5.sup.b) Cl Cl 3.4-3.6(m)8H 5.06(s)2H 6.8-7.4(m)8H Di CH.sub.3
Cl Cl 3.2-3.5(m)8H 5.06(s)2H 6.9-7.4(m)7H 2.2(s)3H;
2.1(s)3HCH.sub.3 (R5.sup.a = R5.sup.b = CH.sub.3 (R5.sup.a and
R5.sup.b Cl Cl 3.4-3.7(m)8H 5.3(s)2H 7.1-8.3(m)11H together
represent CHCHCHCH
__________________________________________________________________________
TABLE 6 ______________________________________ ##STR20## R5.sup.a-b
m.p. of HCl salt (.degree.C.)
______________________________________ Pr.sup.i (R5.sup.a) 124-7 Me
(R5.sup.a) 164-7 Me (R5.sup.b) 122-4 Cl (R5.sup.b) 156-8 F
(R5.sup.a) 123-5 (R5.sup.a = R5.sup.b = CH.sub.3) di-CH.sub.3 144-6
(R.sup.5 and R5.sup.b 180-4 together represent CHCHCHCH Cl
(R.sup.5a) 119-121 ______________________________________
TABLE 7 ______________________________________ ##STR21## R.sup.5a-b
ClCH.sub.2 CH.sub.2 N aromatics other
______________________________________ CH.sub.3 (R5.sup.a)
3.57(m)4H; 3.35(m)4H 7.2-8.4(m)7H 2.32(s)3HCH.sub.3 Pr.sup.1
(R.sup.5a) 3.5(m)4H; 3.34(m)4H 7.2-8.4(m)7H 3.7(m)1HCH;
1.1(d)2H(CH.sub.3) CH.sub.3 (R.sup.5b) 3.7(m)4H; 3.32(m)4H
6.7-8.4(m)7H 2.2(s)3HCH.sub.3 3 F 3.5-3.7(m)8H 7.2-8.1(m)7H 1,4
Naphth 3.5-3.5(m)8H 7.2-8.4(m)10H 2 Cl 3.75(m)8H 6.7-8.4m(7H)
______________________________________
TABLE 8
__________________________________________________________________________
##STR22## R.sup.5a-b ClCH.sub.2 CH.sub.2 N Aromatics ArCH.sub.2 O
.alpha.CH CH.sub.2 CH.sub.2 other
__________________________________________________________________________
CH.sub.3 (R.sup.5a) 3.42(m)8H 7.35-6.9(m)13H 5.2(s)2H 4.4(m)1H
2.5-2.1(m)4H 2.3(s).sup.3H CH.sub.3 5.1(s)2H Pr.sup.i (R.sup.5a)
3.5(m)3.3(m)8H 7.35-6.7(m)13H 5.2(s)2H 4.3(m)1H 2.5-2.1(m)4H
3.6(m)1HCH 5.1(s)2H 1.1(d6H 2 .times. CH.sub.3 CH.sub.3 (R.sup.5b)
3.7(m)3.3(m)8H 6.6-7.4(m)13H 5.14(s)2H 4.2(m)1H 2.5-2.1(m)4H
2.1(s)3H 5.09(s)2H CH.sub.3 3 F 3.60(m)8H 6.7-7.4(m)13H 5.13(s)2H
4.2(m)1H 2.5-2.1(m)4H 5.08(s)2H 1,4 Naphthyl 7.1-8.4(m)16H 5.1(s)2H
4.2(m)1H 2.5-2.1(m)4H 5.16(s)2H 2 Cl 3.7(m)8H 6.7-7.4(m)13H
5.09(s)2H 4.2(m)1H 2.5-2.1(m)4H 5.15(s)2H Cl(R5.sup.a)
3.45-3.62(m)8H 6.98-7.36(m)13H 5.09(s)2H 4.2(m)1H 2.1-2.5(m)4H
5.16(s)2H
__________________________________________________________________________
EXAMPLE 16
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic
acid-.gamma.-anilide
A suspension of .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-anilide (2.0 g) in ethyl acetate (50 ml) was
hydrogenated over 10% palladium on carbon (0.15 g) for 4 h. The
catalyst was removed by filtration and the filtrate was evaporated
to dryness under reduced pressure at 35.degree.. The product,
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-anilide (1 in Scheme 6) was obtained as a white
crystalline solid, 1.4 g (83%) m.p. 110.degree..
Elemental analysis--% Expected C=54.8, H=5.22, N=8.71 % Found
C=54.5, H=5.62, N=8.31
The starting material,
.alpha.-benzyl-4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid .gamma.-anilide, was prepared as described below.
Triethylamine (2 mls) was added to a mixture of
alpha-benzyl-p-tosyl-L-glutamic acid .gamma.-anilide (2.8 g) and
0-(4-[N,N-bis(2-chloroethyl)amino]phenyl-0'-(4-nitrophenyl)carbonate
(2.0 g) in dichloromethane (30 mls). The mixture was stirred at
room temperature for 16 hours and the solvents were removed in
vacuo. The residue was chromatographed on silica gel (Merck Art
9385) and eluted with 10% ethyl acetate in dichloromethane to give
.alpha.-benzyl-4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid .gamma.-anilide as a white solid, 1.9 g (64%).
The starting material
0-(4-[N,N-bis(2-chloroethyl)amino]phenyl-0'-(4-nitrophenyl)carbonate
was prepared as described below.
A solution of triethylamine (10 mls) in dichloromethane (10 mls)
was added to a mixture of 4-nitrophenyl-chloroformate (7.25 g) and
4-(N,N-bis(2-chloroethyl)amino)phenol hydrochloride (10 g) in
dichloromethane (100 mls) over a period of two hours. After
stirring at room temperature for 16 h the solvents were removed
under reduced pressure and the residue was chromatographed on
silica gel (Merck Art. 9385). Elution With dichloromethane and
evaporation of the eluates yielded the product as a red oil.
Trituration with hexane gave a yellow solid which was
recrystallised from benzene/hexane to give
0-(4-[N,N-bis(2-chloroethyl)amino)phenol-0'-(4-nitrophenyl)carbonate
as orange crystals, 10.4 g (71%). m.p. 68.degree..
The starting material p-tosyl-.alpha.-benzyl-L-glutamic acid
.gamma.-anilide was prepared as described below.
N-t-BOC-L-glutamic acid .alpha.-benzyl ester (10 g) and
dicyclohexylcarbodiimide (6.1 g) were dissolved in dichloromethane
(120 mls) and stirred at room temperature for 10 minutes. Aniline
(2.8 mls) was added and the mixture stirred at room temperature for
16 hours. The mixture was filtered and the precipitate washed with
dichloromethane (2.times.15 mls). The filtrate was washed
successively with saturated NaHCO.sub.3 solution (2.times.100 mls)
and water (100 mls) then evaporated. The resulting solid was
recrystallised from EtOAc/hexane to give colourless plates, 8.2 g
(67%). The .gamma.-anilide (12.0 g) and p-toluenesulphonic acid
(5.4 g) in benzene (300 mls) were refluxed for 40 minutes and
allowed to cool overnight. The precipitate was filtered, dried at
the pump and recrystallised from EtOAc/MeOH to give colourless
plates of tosyl-.alpha.-benzyl-L-glutamic acid .gamma.-anilide, 8.2
g (58%).
EXAMPLE 17
N-(4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic
acid-.gamma.-t-butylamide
The process described in Example 16 was repeated using
.alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-t-butylamide in place of .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-anilide to obtain
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-t-butylamide (2 in Scheme 6), which was recrystallised
from ethyl acetate/hexane to give colourless crystals, m.p.
129.degree..
Elemental Analysis--% Expected C=51.9, H=6.32, N=9.09 % Found
C=52.1, H=6.33, N=8.96
The .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-t-b utylamide was obtained in an analogous manner to
that described in Example 16 for the .gamma.-anilide
derivative.
EXAMPLE 18
N-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenylcarbamoyl)-L-glutamic
acid
A solution of di-t-butyl
4-[N,N-bis(2-chloroethyl)amino]-3-methylphenylcarbamoyl-L-glutamate
(0.6 g) in dichloromethane (6 ml) was cooled to 0.degree. C.,
trifluoroacetic acid (15 ml) was added. This solution was then left
at 0.degree. C. for 3 days. The solution was then evaporated to
dryness to yield
(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenylcarbamoyl-L-glutamic
acid as an oil. Yield 0.49 g.
NMR: 8.46(s)1H); 7.15(m)3H; 6.4(d)1H; 4.18(m)1H; 3.55(m)4H;
3.35(m)4H; 2.3(m)2H; 2.23(s)3H, 2.0(m)2H.
The starting material di t-butyl
4-[N,N-bis(2-chloroethyl)-amino]-3-methylphenylcarbamoyl-L-glutamate
was prepared as described below:
Potassium carbonate (27.5 g) was added to a solution of
3-methyl-4-nitroaniline (Journal of Organic Chemistry 33, 3498
(1968) (7.6 g) in ethyl acetate (150 ml), followed by dropwise
addition of a 1.9M solution of phosgene in toluene (27.5 ml)
maintaining the temperature below 30.degree. C. The mixture was
then stirred at ambient temperature for 1 hour. L-glutamic acid
di-t-butyl ester (13 g) was added to the mixture which was then
stirred at ambient temperature overnight. The solution was then
filtered, washed with water, and the organic layer dried over
MgSO.sub.4 and evaporated to an oil. This was then chromatographed
on silica, eluted with Hexane: ethyl acetate 3:1 to give di
t-butyl(3-methyl-4-nitrophenylcarbamoyl-L-glutamate as an oil.
Yield=11.19 g 51%).
NMR: 9.16(s)1H; 8.0(d)1H; 7.43(m)2H; 6.70(d)1H; 4.1(m)1H,
2.51(s)3H; 2.25(m)2H; 1.8(m)2H; 1.41(d)8H.
A solution of di t-butyl
3-methyl-4-nitrophenyl-carbamoyl-L-glutamate (4.7 g) in ethyl
acetate (125 ml) was hydrogenated over 30% Pd/C (0.5 g). The
mixture obtained was then filtered through Celite (a purified and
calcined diatomaceous earth-particle size 20-45 um obtainable inter
alia from Fluka Chemicals Ltd) and evaporated to a dark solid. This
solid was chromatographed on silica eluted using ethyl
acetate:hexane (1:1) to give di t-butyl
3-methyl-4-aminophenylcarbamoyl-L-glutamate as in oil yield=3.71 g
83%.
NMR: 7.98(s)1H; 6.9(m)2H; 6.52(d)1H; 6.15(d)1H; 4.53(s)2H;
4.15(m)1H; 2.25(m)2H; 2.04(s)3H; 1.8(m)2H; 1.45(d)18H.
Ethylene oxide (4.8 g) was bubbled through a solution of di t-butyl
3-methyl-4-aminophenylcarbamoyl-L-glutamate (5 g) in glacial acetic
acid (25 ml) and H.sub.2 O (25 ml). The solution was then stirred
at ambient temperature for 24 hours. After evaporation to dryness,
the residue was redissolved in ethyl acetate, washed with water,
the organic layer dried over MgSO.sub.4 and evaporated to yield di
t-butyl
4-[N,N-bis(2-hydroxyethyl)amino]-3-methylphenylcarbamoyl-L-glutamate
which was used without further purification.
NMR: 8.35(s)1H; 7.1(m)3H; 6.35(d)1H; 4.15(m)1H; 3.35(m)4H;
3.05(m)4H; 2.25(m)2H; 2.20(s)3H; 1.8(m)2H; 1.45(d)18H.
Methanesulfonyl chloride (3.8 ml) was added dropwise to a solution
of di t-butyl
4-[N,N-bis(2-hydroxyethyl)amino]-3-methylphenylcarbamoyl-L-glutamate
(4 g) in pyridine (60 ml) under an argon atmosphere maintaining the
temperature below 30.degree. C. After addition, the solution was
stirred at 80.degree. C. for 2 hours. The solution was cooled and
poured onto 10% citric acid (500 ml), extracted with ethyl acetate,
washed with water, the organic layer dried over MgSO.sub.4 and then
evaporated to a brown oil. This oil was chromatographed on silica
eluted using 5:1 hexane:ethyl acetate to give di t-butyl
4-[N,N-bis(2-chloroethyl)amino[-3-methylphenylcarbamoyl-L-glutamate
as an oil. Yield=7.23 g. 28%.
NMR: 8.42(s)1H; 7.1(m)3H; 6.36(d)1H; 4.13(m)1H; 3.50(m)4H;
3.31(m)4H); 2.3(m)2H; 2.23(s)3H; 1.9(m)2H; 1.4(s)9H; 1.35(s)9H.
The reaction sequence for this Example appears in Scheme 7.
EXAMPLE 19
N-(4-[N,N-bis(2-chloroethyl)amino]benzylcarbonyl)-L-glutamic
acid
Di t-butyl
4-[N,N-bis(2-chloroethyl)amino]benzylcarbonyl-L-glutamate (0.5 g)
was dissolved in CH.sub.2 Cl.sub.2 (1.5 ml) and trifluoroacetic
acid (1.5 ml) was added. The solution was stirred at ambient
temperature for 2 hours. The solution was then evaporated to give
the title compound as an oil (0.8 g).
NMR: 8.22(d)1H; 7.10(d)2H; 6.66(d)2H; 4.19(m)1H; 3.69(s)8H;
2.24(m)2H; 1.9(m)2H; 3.32(s)2H.
The starting material di t-butyl
4-[N,N-bis(2-chloroethyl)amino]benzylcarbonyl-L-glutamate was
prepared as described below:
1-Hydroxybenzotriazole (4.05 g) was added to a solution of
4-nitrophenyl acetic acid (5.4 g) in dimethylformamide (75 ml).
L-Glutamic acid di tert-butyl ester (7.77 g) and then
dicyclohexylcarbodiimide (6.2 g) were added to the mixture. The
mixture was then stirred for 18 hr at ambient temperature. The
mixture was filtered and the filtrate washed with saturated sodium
bicarbonate solution, water and 0.5 m hydrochloric acid and then
dried and evaporated to dryness. The residue was chromatographed on
silica gel by eluting with hexane/ethyl acetate (2:1) to give di
t-butyl 4-nitrobenzylcarbonyl-L-glutamate as an oil 9.7 g
(77%).
NMR: 8.2(d)2H; 7.45(d)2H; 6.45(d)1H; 4.45(m)1H; 3.15(s)2H;
2.1(m)4H; 1.4(d)9H; 1.35(s)9H.
A solution of di t-butyl 4-nitrobenzylcarbonyl-L-glutamate (9.7 g)
in ethyl acetate (200 ml) was hydrogenated over 30% Pd/C (900 mg).
The mixture was then filtered through Celite and evaporated to
yield a yellow oil (di t-butyl 4-aminobenzylcarbonyl-L-glutamate)
which was used without further purification.
NMR: 8.23(d)1H; 6.92(d)2H; 6.50(d)2H; 4.86(s)2H; 4.15(m)1H;
3.24(s)2H; 2.22(m)2H; 1.8(m)2H); 1.4(d)18H. Yield 8.2 g 90%.
Ethylene oxide (7.1 g) was added to a solution of di t-butyl
4-aminobenzylcarbonyl-L-glutamate (8.2 g) in glacial acetic acid
(40 ml) and H.sub.2 O (40 ml). The mixture was then stirred at
ambient temperature for 24 hours. The solution was evaporated to
dryness redissolved in ether, washed with H.sub.2 O, the organic
layer dried over MgSO.sub.4 and evaporates to an oil, (di t-butyl
4-[N,N-bis(2-hydroxyethyl)amino]benzylcarbonyl-L-glutamate) (6.3 g)
which was used without further purification. (6.3 g).
NMR: 8.16(d)1H; 7.02(d)2H; 6.58(d)2H; 4.1(m)1H; 3.4(m)8H;
3.25(s)2H; 2.25(m)2H; 1.8(m)2H; 1.4(s)18H.
Methanesulfonyl chloride (2.91 ml) was added dropwise to a solution
of di t-butyl
4-[N,N-bis(2-hydroxyethyl)amino]benzylcarbonyl-L-glutamate (2.88 g)
in pyridine (45 ml) under an argon atmosphere, maintaining the
temperature below 25.degree. C. After the addition the solution was
stirred at 80.degree. C. for 1 hour. The solution was then cooled
and poured onto 10% citric acid (500 ml) extracted into ether,
washed with water, the organic layer was dried over MgSO.sub.4 and
then evaporated to a brown oil. This oil was chromatographed on
silica eluting with hexane: ethyl acetate 2:1 to give di t-butyl
4-[N,N-bis(2-chloroethyl) amino]benzylcarbonyl-L-glutamate as an
oil (1.3 g) (42%).
NMR: 8.19(d)1H; 7.10(d)2H; 6.66(d)2H; 4.10(m)1H; 3.69(s)8H;
3.32(s)2H; 2.21(m)2H; 1.9(m)2H; 1.37(d)18H. Yield 1.32 g 42%.
The reaction sequence for this Example appears in Scheme 8.
EXAMPLE 20
N-(4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic
acid
A solution of di t-butyl
4-[N,N-bis-(2-chloroethyl)-amino]phenylcarbamoyl-L-glutamate
(prepared as described in Example 3) (500 mg) in 98% formic acid
(10 ml) was allowed to stand at ambient temperature for 24 h. The
solution was evaporated to dryness and the residue chromatographed
on Merck silica gel Art 9385 in dichloromethane/ethyl
acetate/formic acid (7:2:1) to give the title compound as an oil
which crystallized. m.p.=117.degree.-9.degree. C.
EXAMPLE 21
N-(4-[N,N-bis(2-bromoethyl)amino]-3-fluorophenylcarbamoyl)-L-glutamic
acid
A solution of dibenzyl
4[N,N-bis(2-bromoethyl)amino-3-fluorophenyl-carbamoyl-L-glutamate
(0.5 g) in ethyl acetate (10 ml) and 30% Pd/C (100 mg) was stirred
under an atmosphere of hydrogen for 6 hr. The catalyst was filtered
off and the filtrate evaporated to an oil to give the title
compound.
NMR: 8.6(s)1H; 7.35(dd)1H; 7.1-6.8(m)2H; 6.5(d)1H; 4.2(m)1H;
3.7-3.2(m)8H; 2.4-1.6(m)4H.
The starting material for this reaction was prepared in an
analogous manner to that set out in Example 5 but thionyl bromide
was used in place of thionyl chloride.
TABLE 9 ______________________________________ ##STR23## Ex No X R
M.pt ______________________________________ 22 Cl F 111-114.degree.
C. 23 Cl Cl oil 24 Cl CN 105-7.degree. C.
______________________________________
The NMR data for the compound of Example 23 is as follows:
.delta.8.6(broad)1H, .delta.7.6(m)1H, .delta.7.2(m)2H,
.delta.4.25(m)1H; .delta.3.6-3.3(m)8H; .delta.2.4-1.7(m)4H.
The starting materials and intermediates used in the preparation of
the compounds of Examples 21-24 and their properties are listed in
Tables 10-13 below:
TABLE 10 ______________________________________ ##STR24## R m.p
______________________________________ F 99-101 Cl Oil CN 151-4
______________________________________ *NMR(DMSOd.sub.6) 8.15(d)1H:
8.05(q)1H; 7.3(d)1H; 4.65(t)2H(OH); 3.5(m)8H
TABLE 11 ______________________________________ ##STR25## X R m.p.
______________________________________ Cl F 66-8.degree. C. Cl Cl
Oil* Cl CN 106-9 Br F 66-68.degree. C.
______________________________________ *NMR(CDCl.sub.3): 8.3(d)1H;
8.1(q)1H; 7.25(d)1H; 3.75(t)4H; 3.6(t)4H.
TABLE 12 ______________________________________ ##STR26## X R m.p.
(of oxalate salt) ______________________________________ Cl F
146-8.degree. C. Cl Cl 118-21.degree. C. Cl CN 112-6.degree. C. Br
F 134-6.degree. C. ______________________________________
TABLE 13 ______________________________________ ##STR27## X R m.p.
______________________________________ Cl F 81-4.degree. C. Cl Cl
Oil* Cl CN Oil** Br F Oil*** ______________________________________
*NMR(CDCl.sub.3): 7.4(m)1H; 7.3(m)10H; 7.1(m)2H; 5.2(s)2H;
5.05(s)2H; 4.6(m)1H; 3.5-3.4(m)8H; 2.6-2.0(m)4H. **NMR(CDCl.sub.3):
8.25(s)1H; 7.6(m)2H); 7.4-7.2(m)10H); 7.1(d)1H; 6.2(d)1H(NH);
5.2(s)2H); 5.1(s)2H; 4.6(m)1H; 3.7-3.5(m)8H; 2.7-2.2(m)4H. ***NMR:
7.4-6.8(m)13H; 5.7(d)1H; 5.2(s)2H; 5.1(s)2H; 4.6(m)1H;
3.7-3.3(m)8H; 2.6-1.9(m)4H.
EXAMPLES 25-32
The following compounds:
TABLE 14 ______________________________________ ##STR28## Ex No Z
______________________________________ 25 ##STR29## 26 ##STR30## 27
##STR31## 28 ##STR32## 29 ##STR33## 30 ##STR34## 31 ##STR35## 32
##STR36## ______________________________________
were prepared in an analogous manner to that described in Example 1
except as described hereinunder.
The NMR data for each of the compounds of Examples 25-32 is set out
in the following table:
TABLE 15 ______________________________________ Ex No ClCH.sub.2
CH.sub.2 N-- Aromatics .alpha.CH CH.sub.2 --X Other
______________________________________ 25 3.69(m)8H 6.7-6.9 3.97
1.79-1.99 2.2(t)2H (m)4H (m)1H (m)2H 6.7(bs)1H 7.27(bs)1H
7.89(bs)1H 26 3.70(s)8H 6.70-6.93 4.0 1.85-2.2 2.42(m)2H (-)4H
(m)1H (m)2H 3.19(s)3H 7.90(d)1H 27 3.71(s)8H 6.69-7.38 4.04 1.9-2.1
2.45(m)2H (m)8H (m)1H (m)2H 2.26(s)3H 7.89(d)1H 9.83(s)1H 28
3.73(s)8H 6.7-6.9 4.08 2.1-2.27 3.03(t)2H (m)4H (m)1H (m)2H
8.03(d)1H 12.79(bs)1H 29 3.69(s)8H 6.72-6.93 4.38 3.46-3.8
8.12(d)1H (m)4H (m)1H (m)2H 30 3.70(s)8H 6.6-7.6 4.05 1.8-2.3
2.45(m)2H (m)8H (m)1H (m)2H 3.50(s)2H 7.9(d)1H 9.9(s)1H 12.5(broad
s) 31* 3.67(s)8H 6.5-8.5 4.10 1.8-2.3 2.5(m)2H (m)8H (m)1H (m)2H 32
3.70(s)8H 6.22-7.21 4.03 1.8-2.2 2.50(m)2H (m) (m)1H (m)2H
7.79(d)1H 9.52(s)1H ______________________________________ *NMR run
with CD.sub.3 CO.sub.2 D added
The compound of Example 25 was prepared as described in Example 1,
but using
.alpha.-benzyl-4-[N,N-bis(2-chloroethyl)-amino]phenoxycarbonyl-L-glu
tamine in the hydrogenation reaction, the preparation of which is
described hereinafter. Tetrahydrofuran was added to the ethyl
acetate in the hydrogenation reaction to help solubilise the
above-mentioned .alpha.-benzyl compound.
The compounds of Examples 26 and 27 were prepared as described in
Example 1 by hydrogenolysis of the intermediates .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-mesylglutamine and
.alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-(-3-methylphenyl)glutamin
e respectively.
The compound of Example 28 was prepared as described in Example 1
by hydrogenation of benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-(5-tetrazolyl)-.alp
ha.- amino-L-butyrate. The compound of Example 28 was also prepared
by a second method as set out immediately hereinafter. A process
analogous to that described in Example 1 was followed but using
(S)-2-amino-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid (Z. Grzonka et
al. Tetrahedron, 33: 2299-2302, 1977) instead of L-glutamic acid
dibenzyl ester in the reaction with
0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0.sup.1
-(4-nitrophenyl)-carbonate. The reaction was carried out in dry DMF
with 2 equivalents of triethylamine for 20 hours at 25.degree. C.
After evaporation to dryness, the residue was dissolved in ethyl
acetate, washed with dilute citric acid, dried and evaporated to
dryness. The product crystallised slowly from ethyl acetate. The
residue was recrystallised from ethyl acetate to give the compound
of Example 28 (m.p. 173.degree.-5.degree. C.).
The compound of Example 29 was prepared by a procedure similar to
that described for the first preparation of Example 28, but
1-{4-[N,N-bis(2-chloroethyl)amino]phenoxy-carbonylamino}-1-benzyloxy-carbo
nyl-2-(5-thiotetrazole)-ethane was used instead of benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-(5-tetrazolyl)-.alp
ha.-amino-L-butyrate. A similar reaction work up gave a gum which
was purified by column chromatography on silica, eluting with 4%
formic acid/ethyl acetate (by volume).
The compound of Example 29 was also prepared by a second method as
set out immediately hereinafter.
1-amino-1-carboxy-2-(5-thiotetrazole)ethane (600 mg, 2.89 mM) was
suspended in dry DMF (48 mls), triethylamine (0.806 mls, 578 mM)
was added and the suspension stirred.
0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0.sup.1
-(4-nitrophenyl)carbonate (1.10 g, 2.89 mM) was added in a single
portion as a solid and the solution stirred for 20 hours at room
temperature. DMF was removed in vacuo. The residue was dissolved in
ethyl acetate and dilute citric acid. The ethyl acetate layer was
washed with water, dried with sodium sulphate, filtered and
evaporated. The crude product obtained was chromatographed on
silica gel and eluted with 4% formic acid in ethyl acetate to give
4-[N-N,bis(2-chloroethyl)amino]phenoxycarbonyl
amino-1-carboxy-2-(5-thiotetrazole)ethane as a glassy solid (0.963
g).
NMR (DMSOd.sub.6) 3.46(dd, 1H); 3.69(s, 8H); 3.8(dd, 1H); 4.38(m,
1H); 6.72(d, 2H); 6.93 (d, 2H); 8.12 (d, 1H).
The compounds of Examples 30 and 31 were prepared as described in
Example 1 by hydrogenation of .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-[3-(benzyloxycarbon
yl-methyl)-phenyl]-L-glutamine and .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.[[3-(5-tetrazolyl)-p
henyl]-L-glutamine respectively.
The compound of Example 32 was prepared as described in Example 1
by hydrogenation of
.alpha.-benzyl-4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-[3-(
benzyloxycarbonylamino)phenyl]-L-glutamine.
Intermediates for Use in Preparing the Compounds of Examples
25-32
.alpha.-Benzyl-4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamine,
for use in preparing the compound of Example 25, was prepared as
described in Example 1 from
0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0'-(4-nitrophenyl)carbonate
but substituting .alpha.-benzyl-L-glutamine (L. Zervas et al. J.
Am. Chem. Soc 87 (1), 99-104, 1965) for L-glutamic acid dibenzyl
ester tosylate. The product was purified by flash column
chromatography on silica, elution being with 80% EtOAc/20% Hexane.
The product was obtained as a white solid after trituration with
ether.
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-mesyl
L-glutamine, for use in the preparation of the compound of Example
26, was prepared as follows:
N-Boc-.alpha.-benzyl-L-glutamate [E. Klieger et al, Ann. 673.
196-207, 1964] (10 g) in 50 ml dry dichloromethane was treated with
dimethylaminopyridine (DMAP)(3.9 g) and dicyclohexylcarbodiimide
(6.73 g). Methane sulphonamide (3.04 g) was then introduced to the
reaction flask and the reaction continued at 25.degree. C. for 20
hrs. Dichloromethane was evaporated and the residue redissolved in
ethyl acetate. Ethyl acetate solution was then washed with 0.25M
citric acid, followed by water and then dried over Na.sub.2
SO.sub.4 (anhydrous). Evaporation of the ethyl acetate extract gave
a residue which was purified on silica using flash column
chromatography using as eluent methylene chloride, then 5%
methanol/methylene chloride and then 10% methanol/methylene
chloride to give the Boc-protected acyl sulphonamide: ##STR37##
NMR: .delta. 1.37(s)9H; 1.98(m)2H; 2.34(t)2H; 5.11(d)2H; 7.26(d)1H;
7.36(s)5H; 11.64(s)1H.
3.6 g of the Boc-protected acyl sulphonamide was suspended in 50 ml
ethyl acetate followed by the addition of 8 equivalents of HCl
saturated ethyl acetate (22.4 ml of 3.1M solution). The starting
material sulphonamide went into solution and the reaction was
allowed to continue for 20 hrs, under argon, at 25.degree. C.
.alpha.-Benzyl .gamma.-mesyl-L-glutamine hydrochloride of the
formula: ##STR38## was produced as a white solid which was filtered
off and washed with anhydrous ether before drying in a
dessicator.
NMR: .delta.2.08(m)2H; 2.52(m)2H; 3.20(s)3H; 4.09(t)1H;
5.15-5.35(dd)2H, 7.39-7.50(m)5H, about 9 (broad) 3H.
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-mesylglutamine was
prepared as described in Example 1 from
0-(4-N,N-bis(2-chloroethyl)amino]-phenyl)-0'-(4-nitrophenyl)carbonate
but substituting .alpha.-benzyl .gamma.-mesyl-L-glutamine
hydrochloride for L-glutamic acid dibenzyl ester tosylate. The
product of the reaction was purified by column chromatography on
silica, eluting with 10% formic acid in ethyl acetate (by
volume).
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-(3-methylphenyl)-L-
glutamine, for use in the preparation of the compound of Example 27
was prepared as follows:
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamate was
prepared as described in Example 1 from
0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0'-(4-nitrophenyl)carbonate
but substituting .alpha.-benzyl-L-glutamate (Ref. C.
Coutsogeorgopoulos et al J. Am. Chem. Soc. 83, 1885, 1961) for
L-glutamic acid dibenzyl ester. Moreover the reaction was performed
in dry DMF at 25.degree. C. for 2 hrs. The product was purified by
column chromatography (silica, Merck Art 9385) using ethyl
acetate/hexane mixtures in the range 70/30 respectively to 100%
ethyl acetate. The NMR data for the product is set out in Table
17.
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamate (300 mg)
in dry tetrahydrofuran (5 ml) was treated with 1.1 equivalents of
triethylamine. Isobutylchloroformate (0.08 ml, 1.1 equiv) in dry
THF (10 ml) was added slowly to the reaction mixture at -25.degree.
C. After 15 minutes, 1.1 equivalents (0.08 ml) m-toluidine in dry
tetrahydrofuran (5 ml) was added. The mixture was allowed to warm
up to 25.degree. C. and was then continuously stirred for 18 hrs.
The reaction mixture was then filtered and the filtrate evaporated
to give a residue which was purified by flash column chromatography
using mixtures of ethyl acetate/hexane as eluent. Evaporation of
appropriate fractions yielded .alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-(3-methylphenyl)-L-
glutamine as a white crystalline solid. The NMR data from this
product is set out in Table 17.
1-{4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonylamino}-1-benzyloxycarbonyl
-2-(5-thiotetrazole)-ethane for use in the prepartion of the
compound of Example 29 was prepared as follows:
1-amino-1-carboxy-2-(5-thiotetrazole)ethane was prepared by adding
.beta.-chloroalanine (18.50 g, 115.6 mmol) (Sigma Chemical Co) and
5-thiotetrazole (11.79 g, 115.6 mmol (prepared as described in
European Patent Publication No. 33965) to 230 mls of a 2M sodium
hydroxide solution with stirring. The reaction mixture was then
heated from room temperature to 90.degree. C. for 11/2 hours. The
reaction mixture was then allowed to cool to room temperature and
then cooled further with ice/methanol bath cooling, the mixture was
acidified to pH4.0.with concentrated hydrochloric acid and stirring
was maintained for 1/2 hour. The resulting precipitate was filtered
off, washed with cold water and then with ether and filtered dry
with suction to give 6.9 g. The mother liquor's pH was re-checked
and found to have risen to about 5.5. The above procedure was
repeated and a further 2.3 g of product obtained. This was dried
under high vacuum.
NMR: .delta. 3.30-3.50 ppm(m)2H; 4.16-4.22 ppm(q)1H; 7.47 ppm
(broad) H.sub.2 O exchanging with NH.sub.2
Elemental Analysts--Expected C=23.2, H=4.4; N=33.8 (+1 mol of
H.sub.2 O) Found C=23.1; H=4.4; N=33.7 (9.6% H.sub.2 O)
1-{4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonylamino}-1-benzyloxycarbonyl
-2-(5-thiotetrazole)-ethane was prepared as described in Example 1
from
0-(4-[N,N-bis(2-chloroethyl)amino]-phenyl)-0'-(4-nitrophenyl)carbonate,
but substituting
.gamma.-(5-thiotetrazolyl)-.alpha.-benzyloxycarbonyl-amino-L-butyric
acid (Ref. Z Grzonka et al Tetrahedron Letters 33 2399-2302, 1977)
for L-glutamic acid dibenzyl ester. Dry dimethylformamide was used
as solvent and the reaction was carried out at 25.degree. C. The
reaction mixture worked up after 2 hours and the product purified
by flash column chromatography on silica and eluting with a mixture
made up of 2% formic acid in ethyl acetate/methylene chloride (3:1
by volume). The NMR data for this product is set out in Table
16.
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-[3-(benzyloxycarbon
ylmethyl)phenyl]-L-glutamine, for use in Example 27 was prepared as
follows:
3-amino-phenylacetic acid benzyl ester p-toluene sulphonic acid was
prepared by adding 3-aminophenylacetic acid (10 g) and p-toluene
sulphonic acid monohydrate (13.2 g) to benzyl alcohol (27.2 ml) in
toluene (30 ml). The mixture was heated under reflux and the water
formed collected in a Dean-Stark receiver. When all the water had
been distilled off the mixture was allowed to cool to 25.degree. C.
before diluting with diethyl ether and placing in an ice-bath for 1
hr. The crystalline p-toluene sulphonate was filtered off and dried
in a dissicator (23.5 g)
NMR .delta. 2.31(s)3H, 3.82(s)2H, 5.11(s)2H, 7.11(d)2H,
7.25-7.45(m)8H, 7.52(d)2H.
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-[3-(benzyloxycarbon
ylmethyl)phenyl]-L-glutamine was prepared as described in Example
27 but substituting 3-aminophenylacetic acid benzyl ester for
m-toluidine.
.alpha.-Benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxy-carbonyl-.gamma.-[3-(5-tetrazolyl)p
henyl]-L-glutamine, for use in Example 31 was prepared as described
in Example 27, but substituting 3-(tetrazol-5-yl)-aniline for
m-toluidine. The 3-(tetrazol-5-yl)aniline required as a starting
material was prepared as follows. To a solution of
5-(3-nitrophenyl) tetrazole (Finnegan W. G.; Henry R. A., and
Lolquist R. J.A.C.S. 80:3908 (1958)) (52 g) in ethanol (2.51) was
added 10% palladium on carbon (5 g) and the mixture stirred under
an atmosphere of hydrogen for 16 h. The catalyst was removed by
filtration and the filtrate evaporated to give the desired starting
material (40.5 g; m.p.=188.degree.-9.degree. C.).
.alpha.-Benzyl-4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-.gamma.-[3-(b
enzyloxycarbonylamino)phenyl]-L-glutamine for use in Example 32 was
prepared as described in Example 27 but substituting
3-(benzoyloxycarbonylamino)aniline for m-toluidine.
NMR ClCH.sub.2 CH.sub.2 N: 3.68(m)8H Aromatics: 6.68-7.74(m)1H
ArCH.sub.2 O: 5.12(s)2H; 5.14(s)2H .alpha.CH: 4.18(m)1H Other:
1.9-2.15(m)2H; 2.45(m)2H; 8.12(d)1H; 9.7(s)1H; 9.9(s)1H.
3-(benzoyloxycarbonylamino)aniline used as an intermediate in
preparation of Example 32, was prepared as follows. To a cold
solution (0.degree.) of m-phenylene diamine (10 g) in ethyl acetate
(200 ml) was added aq KHCO.sub.3 (9 g in 300 ml water) with
stirring. Benzyl chloroformate (13.2 ml) in ethyl acetate (100 ml)
was added dropwise over ten minutes, stirred at 0.degree. C. for
one hour and then made acid (pH2) by addtion of M.HCl (aq). The
product was extracted into ethyl acetate (250 ml), washed with
brine, dried over MgSO.sub.4 and reduced in "vacuo" to give an oil.
The oil was chromatographed on Merck silica gel Art 9385 and eluted
with hexane/ethyl acetate (7:3) to give 8 g of the intermediate as
a low melting point solid (36% yield).
NMR: 9.32(s)1H; 7.30(m)5H; 6.80(t)1H; 6.70(m)1H; 6.50(m)1H;
6.12(m)1H; 5.0(s)2H; 4.88(s)2H.
TABLE 16
__________________________________________________________________________
X ClCH.sub.2 CH.sub.2 N-- Aromatics ArCH.sub.2 O .alpha. CH
CH.sub.2 --X Other
__________________________________________________________________________
CH.sub.2 CO.sub.2 H 3.63(m)8H 6.62-7.31(m)9H 5.11(s)2H 4.14(m)1H
1.8-2.05(m)2H 7.89(d)1H 2.35(m)2H CH.sub.2 CONH.sub.2 3.71(m)8H
6.7-7.3(m)9H 5.15(s)2H 4.13(m)1H 1.8-2.03(m)2H 2.23(t)2H 6.77(s)1H
7.28(s)1H 8.08(d)1H CH.sub.2 CONHSO.sub.2 Me 3.71(m)8H
6.7-7.37(m)9H 5.15(s)2H 4.15(m)1H 1.8-2.1(m)2H 2.49(m)2H 3.19(s)3H
11.7(bs)1H 8.1(d)1H CH.sub.2 CONH-- 3.71(m)8H 6.69-7.42(m)13H
5.18(s)1H 4.19(m)1H 1.9-2.2(m)2H 2.49(m)2H 2.25(s)3H 8.11(d)1H
9.83(s)1H CH.sub.2 -- 3.71(m)8H 6.7-7.37(m)9H 5.17(s)2H 4.23(m)1H
2.1-2.3(m)2H 8.23(m)2H 3.03(t)2H CH.sub.2 CONH-- 3.70(m)8H
6.6-7.6(m)18H 5.10(s)2H 4.19(m)1H 1.9-2.3(m)2H 2.45(m)2H 5.17(s)2H
3.67(s)2H 8.13(d)1H 9.90(s)1H CH.sub.2 CONH-- 3.70(s)8H
6.6-8.5(m)13 5.15(s)2H 4.2(m)1H 1.9-2.3(m)2H 2.15(m)2H 8.15(d)1H
10.2(s)1H
__________________________________________________________________________
EXAMPLE 33
N-(4-[N,N-bis(2-iodoethyl)amino]phenoxy-carbonyl)-L-glutamic
acid
4-[N,N-bis(2-iodoethyl)amino]phenoxy-carbonyl-L-glutamic acid
di-t-butyl ester (188 mg) (See Scheme 9 - Compound 14) was
suspended in trifluoroacetic acid (TFA) (4 ml) and stirred for 30
min at ambient temperature. TFA was removed under reduced pressure;
the remaining oil was diluted with ethyl acetate (3 ml) and
evaporated to give
4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl-L-glutamic acid-1.4
TFA-0.8 EtOAc (162 mg) 82% yield; (Compound 15 in Scheme 9).
NMR: 1.84-2.01 (m) 1H; 2.36 (m) 2H; 3.31 (t) 4H; 3.72 (t) 4H; 4.02
(m) 1H 6.66 (d) 2H; 6.94 (d) 2H;. 7.92 (d) 1H.
4-[N,N-bis(2-iodoethyl)amino]phenoxy-carbonyl-L-glutamic acid
di-t-butyl ester used as intermediate was prepared as follows:
a) A solution of the product
bis(2-mesyloxyethyl)amino]phenoxycarbonyl-L-glutamic acid
di-t-butyl ester (1.0 g) (See Scheme 9 - compound 11) in
acetonitrile (50 ml) was stirred with sodium iodide (1.0 g) at
70.degree. C. for 20 h. The reaction mixture was filtered and the
filtrate concentrated under vacuum. The residue was chromatographed
on silica gel; eluted with ethyl acetate in cyclohexane (1:5) to
obtain 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl-L-glutamic acid
di-t-butyl ester (see Scheme 9 - Compound 14) as an oil (0.75 g)
68% yield.
NMR: 1.41 (s) 9H; 1.43 (s) 9H; 1.81-1.95 (m) 1H; 2.34 (m) 2H; 3.31
(t) 4H; 3.72 (t) 4H; 4.00 (m) 1H; 6.67 (d) 2H; 6.93 (d) 2H; 7.91
(d) 1H.
b) bis(2-mesyloxyethyl)amino]phenoxycarbonyl-L-glutamic acid
di-t-butyl ester
A solution of
4-[N,N-bis(2-hydroxyethyl)amino]phenoxycarbonyl-L-glutamic acid
di-t-butyl ester (2.53 g) (obtained as described in Example 8--see
also Scheme 4) in pyridine (9 ml) was stirred with methanesulphonyl
chloride (1.8 ml) at 2.degree. C. for 20 min followed by 80.degree.
C. for 11 min. The reaction mixture was partitioned between ethyl
acetate and citric acid/water (10%). The organic phase was
separated, washed with water, dried (Na.sub.2 SO.sub.4) and
evaporated to dryness. The residue was chromatographed on silica
gel; eluted with ethyl acetate in dichloromethane (1:9) to obtain
4-[N,N-bis(2-mesyloxyethyl)amino]phenoxycarbonyl-L-glutamic acid
di-t-butyl ester (Compound 11 in Scheme 9) as an oil (0.95 g) 28%
yield.
NMR: 1.41 (s) 9H; 1.43 (S) 9H; 1.8-1.99 (m) 1H; 2.34 (m) 2H; 3.16
(s) 6H; 3.72 (t) 4H; 3.9 (m) 1H; 4.31 (t) 4H; 6.78 (d) 2H; 6.92 (d)
2H; 7.9 (d) 1H.
EXAMPLE 34
N-(4-[N,N-bis(2-bromoethyl)amino]phenoxy-carbonyl)-L-glutamic
acid
4-[N,N-bis(2-bromoethyl)amino]phenoxy-carbonyl-L-glutamic acid
di-t-butyl ester (133 mg) [see Scheme 9--compound (12)] was
suspended in TFA (4 ml) and stirred for 30 min at ambient
temperature. TFA was removed under reduced pressure; the remaining
oil was diluted with ethyl acetate (3 ml) and evaporated to give
4-[N,N-bis(2-bromoethyl)amino]phenoxy-carbonyl-L-glutamic acid-1.3
TFA-0.9 EtOAc (126 mg) 80% yield. [see Scheme. 9--compound
(13)].
NMR: 1.83-2.01 (m) 1H; 2.36 (m) 2H; 3.58 (t) 4H; 3.76 (t) 4H; 4.03
(m) 1H; 6.71 (d) 2H; 6.94 (d) 2H; 7.92 (d) 1H.
4-[N,N-bis(2-bromoethyl)amino]phenoxy-carbonyl-L-glutamic acid
di-t-butyl ester used as intermediate was prepared as follows:
A solution of bis(2-mesyloxyethyl)amino]phenoxy-carbonyl-L-glutamic
acid di-t-butyl ester (0.48 g) (compound 11 in Scheme 9--obtained
as described in Example 8--see also Scheme 4) in acetonitrile (30
ml) was stirred with lithium bromide (0.26 g) at 70.degree. C. for
22 h. The reaction mixture was filtered and the filtrate
concentrated under vacuum. The residue was chromatographed on
silica gel; eluted with ethyl acetate in dichloromethane (1:5) to
obtain 4-[N,N-bis(2-bromoethyl)amino]phenoxycarbonyl-L-glutamic
acid di-t-butyl ester (compound 12 in Scheme 9) as an oil (0.37 g)
83% yield.
NMR: 1.41 (s) 9H; 1.43 (s) 9H; 1.8-1.98 (m) 1H; 2.34 (m) 2H; 3.58
(t) 4H; 3.76 (t) 4H; 3.97 (m) 1H; 6.72 (d) 2H; 6.93 (d) 2H; 7.93
(d) 1H.
EXAMPLE 35
N-(4-[N,N-bis-(2-chloroethyl)amino]2-fluoro-phenylcarbamoyl)-L-glutamate
The titled compound (NMR (dmso): 8.0(s) 1H; 7.65(t)1H; 6.6(m) 3H;
4.2(m)3H; 3.7(s)8H; 2.28(m)2H; 1.8(m)2H) was prepared from the
intermediate dibenzyl
4-[N,N-bis(2-chloroethyl)amino]2-fluorophenylcarbamoyl-L-glutamate
(NMR (dmso): 7.62(t)1H; 7.35(s)10H; 6.8(d)1H; 6.57(m)2H; 5.1(d)4H;
4.33(m)1H; 3.7(s)8H; 2.43(m)2H; 2.0(m)2H) in a manner analogous to
the corresponding step of Example 5.
The intermediate was prepared as follows:
Ethylene oxide (6.6 g) was added to 3-fluoro-4-nitroaniline (1.3 g)
in glacial acetic acid (30 ml) and the reaction mixture was kept in
a closed flask at laboratory temperature for 72 h. The solution was
evaporated under reduced pressure to half of its original volume,
diluted with saturated aqueous sodium chloride solution and
extracted three times with ethyl acetate. The ethyl acetate
extracts were combined, washed with saturated aqueous sodium
bicarbonate, evaporated, and the residue was purified by flash
column chromatography on silica gel. After elution with hexane
containing 50% (v/v) ethyl acetate to remove unchanged starting
material and monosubstituted product. Elution with ethyl acetate
gave the product 2',2'-(3-fluoro-4-nitroanilino)diethanol (m.pt.
99.degree.-101.degree. C.).
The product so obtained (280 mg) was dissolved in dichloromethane
(7.5 ml), pyridine (0.1 ml) was added and the solution was cooled
in an ice/water bath. Thionyl chloride (0.25 ml) was added
dropwise, with stirring. When the addition was complete the
reaction mixture was heated under reflux for 1 hour and then left
at laboratory temperature for 20 hours. The solution was diluted
with dichloromethane (10 ml) and washed three times with water,
dried over sodium sulphate, evaporated, and the residue was
recrystallised from methanol to give the product
[N,N-bis(2-chloroethyl]-3-fluoro-4-nitroaniline (m.pt
97.degree.-98.degree. C.).
The product so obtained (200 mg) in tetrahydrofuran (7.5 ml) was
stirred for 16 hours in an atmosphere of hydrogen in the presence
of palladium/charcoal (20 mg of 5% w/w). The catalyst was removed
by filtration and the solvent was evaporated. The resulting residue
was dissolved in the minimum volume of methanol and the crude
product was precipitated by addition of excess diethyl ether
saturated with hydrogen chloride. Recrystallisation from
methanol/diethyl ether gave the product
4-[N,N-bis(2-chloroethyl)amino-2-fluoro-anilinium chloride (m.pt.
195.degree.-200.degree. C.,d).
The resulting product yes converted into the desired intermediate
in a manner analogous to the corresponding step in Example 5.
EXAMPLES 36-43
The structures and elemental analysis data for compounds of
Examples 36-43 are set out in Table 17.
The compounds listed in Table 17 were prepared according to the
procedures described in Example 16. Thus the compound of example 36
was prepared by substituting benzyl-4-aminobenzoate (Aldrich
Chemical Co Ltd) for aniline in Example 16. Similarly, the
compounds of examples 37-43 were prepared by substituting benzyl
4-aminobenzoate, sec-butylamine, n-propylamine, iso-propylamine,
cyclohexylamine, benzylamine or p-benzyloxyaniline for aniline in
Example 16.
TABLE 17
__________________________________________________________________________
##STR39## % Expected % Found Ex. No. W C H N C H N
__________________________________________________________________________
36 CONH-p-C.sub.6 H.sub.5 COOH 51.5 5.26 7.83 51.7 5.10 7.50 37
CONH-n-C.sub.4 H.sub.9 51.9 6.32 9.09 52.0 6.38 8.97 38
CONH-sec-C.sub.4 H.sub.9 51.9 6.32 9.09 52.2 6.34 9.09 39
CONH-n-C.sub.3 H.sub.7 50.9 6.07 9.37 50.6 6.35 8.95 40
CONH-i-C.sub.3 H7 50.9 6.07 9.37 51.1 6.07 9.40 41 CONHC.sub.6
H.sub.11 54.1 6.40 8.60 54.3 6.73 8.72 42 COHNCH.sub.2 C.sub.6
H.sub.5 55.6 5.48 8.46 56.0 5.52 8.21 43 CPNH-p-C.sub.6 H.sub.5 OH
53.0 5.06 8.43 53.0 5.24 7.90
__________________________________________________________________________
EXAMPLE 44
N-(4-N,N-bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic
acid
A solution of the intermediate dibenzyl
4-[N,N-bis(2-chloroethyl)amino]-phenylcarbamoyl-L-glutamate (1.138
g) in DMF (15 ml) was hydrogenated over 10% Pd/C for 16 hours.
After filtration and evaporation in vacuo, the residue was
dissolved in CHCl.sub.3 (20 ml). After 18 hours the crystalline
precipitate was filtered off and dried in vacuo to obtain
4-[N,N-bis-(2-chloroethyl)amino]phenylcarbamoyl-L-glutamic acid.
Yield, 730 mg (93%). After recrystallisation from
acetone/CHCl.sub.3 microscopic rods formed m.p.
116.degree.-118.degree. C.
NMR (CD.sub.3 COCD.sub.3): .delta. 8.0(s)1H; 7.2(d)2H; 6.6(d)2H;
6.2(d)2H NH; 4.4(m)1H; 3.6(m)8H; 2.5-1.9(m)4H.
The dibenzyl intermediate was prepared as follows (see Scheme
10).
Dibenzyl glutamate p-tolunesulphonate (available Bachem U.K.; 0.25
g) was dissolved in dry methylene chloride (10 ml) under argon and
cooled to 0.degree. C. Pyridine (0.162 ml) was added, followed
rapidly by phosgene in toluene (1.93M, 0.311 ml). The solution was
stirred at 0.degree. C. for 2 hours, pyridine (0.05 ml) added and
followed by 4-[N,N-bis (chloroethyl)amino]anilinium chloride in one
portion. The mixture was stirred for 10 minutes at 0.degree. C. and
then for 18 hours at room temperature. The reaction mixture was
then diluted with ethyl acetate and water. The organic layer was
then washed in turn with dilute citric acid (2.times.), water and
saturated brine; dried and evaporated to give the desired dibenzyl
intermediate as a solid.
An alternative route to the dibenzyl intermediate is as
follows:
To a solution of triphosgene (1 g) in chloroform (80 ml) was added
at 10.degree. C. 4-[N,N-bis-(2-chloroethyl)amino]anilinium chloride
(2.7 g). Whilst keeping the tempeature at 10.degree. C.,
triethylamine (4.15 ml) was added and the mixture was stirred and
allowed to warm to ambient temperature for 15 minutes. To this
mixture was added in one portion dibenzyl glutamate tosylate and
triethylamine (1.7 ml). After 1.5 hours at ambient tempeature the
mixture was diluted with chloroform (100 ml), washed twice with
water, dried and evaporated to dryness. The resdiue was
chromatographed on Merck silica gell Art 9385, eluting with ethyl
acetate hexane to give dibenzyl
4[N,N-bis(2-chloroethyl)amino]-phenylcarbamoyl-L-glutamate (2.5 g).
m.p. 119.degree.-22.degree. C.
EXAMPLE 45
4-[N,N-bis(2-chloroethyl)amino]phenylcarbamoyl-.gamma.-[N(3-carboxymethyl)a
nilino]-L-glutamate (compound 7 in Scheme 11)
The titled compound was prepared as follows (see scheme 11). A
solution of the intermediate (compound 6 in Scheme 11; 1 g) in 20
THF was hydrogenated over 30% d/c (100 mg) for 4 hours. The mixture
was then filtered through celite and evaporated to yield a brown
oil which was then purified by flash column chromatography using 1%
and 3% formic acid/ethylacetate mixtures as eluent, evaporation of
appropriate fractions and interaction with ether yielded 300 mg of
the titled compound.
NMR: ClCH.sub.2 CH.sub.2 N: 3.66(s)8H; Aromatics: 6.66-7.46(m)8H;
.alpha.CH:4.22(m)1H; Other: 1.66-2.06(m)2H); 2.39(m)2H); 3.49(s)2H;
6.29(d)1H; 8.33(s)1H; 9.93(s)1H; 12.5(b.s)2H.
The intermediate was prepared as follows:
(a) 3-amino-phenylacetic acid benzyl ester p-toluene sulphonic acid
was prepared by adding 3-aminophenylacetic acid (10 g) p-toluene
sulphonic acid monohydrate (13.2 g) to benzyl alchohol (27.2 ml) in
toluene (30 ml). The mixture was heated under reflux and the water
formed collected in a Dean-Stark receiver. When all the water had
been distilled off the mixture was allowed to cool to 25.degree.
C., before diluting with diethyl ether and placing in an ice-bath
for 1 hour. The crystalline p-toluene sulphonate was filtered off
and the product dried in a dessicator (23.5 g) NRM .delta.
2.31(s)3H, 3.82(s)2H, 5.11(s)2H, 7.11(d)2H, 7.25-7.45(m)8H,
7.52(d)2H.
(b) N Boc .alpha. benzyl L glutamate, (5 g) in 20 ml) dry DMF at
5.degree. C. was heated with (1.1 eq; 2.45 g) hydroxybenzothiazole
(HOBT) and the reaction mixture stirred at this temperature under
argon for 10 minutes. (DCCI) Dicyclohexylcarbodimide (1.1 eq; 3.37
g) was then added and the reaction stirred for a further 10 minutes
at 5.degree. C. before allowing to warm to 25.degree. C. and
stirring for a further 45 minutes. 3 amino phenylacetic acid benzyl
ester p-toluene sulphonic acid (the product obtained in (a)) (1.1
eq; 6 g) together with 1.1 eq triethylamine (2.23 ml) in 10 ml dry
DMF was then added and the reaction mixture stirred at 25.degree.
C. for a further 20 hours. The precipitate of dicyclohexylurea was
then filtered off, and the DMF filtrates evaporated to dryness. The
residue was then redissolved in EtoAc. The ethyl acetate solution
was then washed with NaHCO.sub.3 (aq), then brine and then dried
over Na.sub.2 SO.sub.4 (anhydrous). Evaporation of the ethylacetate
extract gave a residue which was then purified by flash column
chromatography using 30,40 and 50% EtoAc/hexane mixtures as eluent.
Evaporation of appropriate fractions yielded 5 g of prudct
(compound 4 in scheme 11).
NMR Data: 1.39(s)9H; 1.81-2.10(m)2H; 2.38(m)2H; 3.69(s)8H;
4.03(m)1H; 5.12(m)4H; 6.92(d)1H; 7.21(m)1H; 7.35(m)11H; 7.42(d)1H;
7.52(s)1H; 9.89(s)1H.
(c) The product obtained in (b) (5 g) was suspended in 10 ml ether
and 5 ml dichloromethane added (to aid solubility) followed by 8 eq
of saturated etheral/HCl. The reaction mixture was then allowed to
stir for 20 hours at 25.degree. C. The product was at this stage an
immiscible oil. The ether was then evaporated and the residue
azeotroped twice with toluene before adding ether and evaporating
down to yield 5 g of product (compound 5 in scheme 11) as a yellow
foam.
NMR Data CDCl.sub.3 : .delta.2.35(m)2H; .delta.2.65(m)2H;
.delta.3.50(s)2H; .delta.4.22(bs)1H; .delta.5.01(s)2H;
.delta.6.8-7.6(m)14H; .delta.8.65(bs)3H; .delta.9.35(s)1H.
(d) 1.1 eq (0.66 g) of 1.1 carbonyldiimidazole in 20 ml dry THF at
5.degree. C. was treated with a solution containing 1 g of 4[N.N
bis (2-chloroethyl)amino]anilinium chloride. The reaction mixture
was then stirred at 5.degree. C. for 15 minutes before adding 1 eq
(1.84 g) of the product obtained in (c) with 1.1 eq (0.56 ml)
triethylamine in 10 ml dry THF, and stirred for a further 2 hours
at 25.degree. C. The triethylamine hydrochloride ppt was then
removed by filtration and the THF filtrates evaporated and the
residue redissolved in EtoAc. The ethyl acetate solution was then
washed with water, followed by 0.25M citric acid, then brine and
dried over Na.sub.2 SO.sub.4 (anhydrous). Evaporation of the ethyl
acetate gave a residue which was purified by flash column
chromatography using 30,40 and 50% ethylacetate/hexane mixture as
eluent. Evaporation of appropriate fractions yielded the desired
intermediate (compound 6 in scheme 11).
NMR Data: ClCH.sub.2 CH.sub.2 N: 365(s)8H; Aromatics:
6.63-7.5(m)18H; ArCH.sub.2 O: 5.10(s)2H; 5.12(s)2H; .alpha.CH;
4.32(m)1H; Other: 1.83-2.13(m)2H; 2.41(m)2H; 6.42(d)1H; 8.25(s)1H;
9.91(s)1H.
EXAMPLE 46
4-{N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-(3,5-dicarboxy)anilide
The process described in Example 16 was repeated using
.alpha.-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-(3,5-dicarboxybenzyl)anilide in place of
.alpha.-benzyl
4-(N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-anilide to obtain
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-(3,5-dicarboxy) anilide as colourless crystals (m.p.
167.degree.-170.degree. C.).
Elemental analysis:--% expected C=49.6 H=4.87 N=6.68 % found C=49.7
H=4.9 N=6.7
The .alpha.-benzyl 4-benzyl
4-[N,N-bis(2-chloroethyl)amino]phenoxycarbonyl-L-glutamic
acid-.gamma.-(3,5-dicarboxybenzyl)anilide was obtained in an
analogous manner to that described in Example 16 for the
.gamma.-anilino derivative. ##STR40##
* * * * *